阅读说明：本技术 无缝核酸装配方法 (Seamless nucleic acid assembly method ) 是由 丽贝卡·纽金特 陈思远 埃利安·李 内森·雷纳德 于 2018-06-12 设计创作，主要内容包括：本文提供了用于无缝核酸装配的方法、系统和组合物。本文提供的方法、系统和组合物提供了核酸的有效装配而无需引物去除。用于无缝核酸装配的方法、系统和组合物包括使用内切核酸酶或外切核酸酶,任选地结合额外的酶,以装配核酸或多核苷酸。(Provided herein are methods, systems, and compositions for seamless nucleic acid assembly. The methods, systems, and compositions provided herein provide for efficient assembly of nucleic acids without primer removal. Methods, systems, and compositions for seamless nucleic acid assembly include the use of endonucleases or exonucleases, optionally in combination with additional enzymes, to assemble nucleic acids or polynucleotides.)

1. A method of nucleic acid assembly comprising:

(a) providing a plurality of polynucleotides, wherein each of said polynucleotides does not comprise terminal regions having sequence homology to another polynucleotide of said plurality; and

(b) mixing the plurality of polynucleotides with an exonuclease, an endonuclease, a polymerase, and a ligase, wherein the plurality of polynucleotides anneal in a continuous predetermined order based on the complementary sequence between adjacent polynucleotides.

2. The method of claim 1, wherein the exonuclease is exonuclease III.

3. The method of claim 1, wherein the endonuclease is a flap endonuclease.

4. The method of claim 3, wherein the flap endonuclease is flap endonuclease 1, exonuclease 1, XPG, Dna2, or GEN 1.

5. The method of claim 1, wherein the polymerase has 5 'to 3' polymerase activity.

6. The method of claim 1, wherein the polymerase is a DNA polymerase.

7. The method of claim 1, wherein the ligase catalyzes the ligation of at least two nucleic acids.

8. The method of claim 4, wherein the concentration of flap endonuclease 1 is in the range of about 0.32U to about 4.8U.

9. The method of claim 2, wherein the concentration of exonuclease III is in the range of about 0.1U to about 10U.

10. The method of claim 5, wherein the concentration of the polymerase is in the range of about 0.01U to about 2U.

11. The method of claim 7, wherein the concentration of the ligase is at most about 2.0U.

12. The method of claim 7, wherein the concentration of the ligase is in the range of about 4.0U to about 8.0U.

13. A method of nucleic acid assembly comprising:

(a) providing a first double-stranded nucleic acid;

(b) providing a second double-stranded nucleic acid;

(c) providing a third double stranded nucleic acid comprising, in 5 'to 3' order: a 5 'flanking adaptor sequence, a first homologous sequence homologous to the first double-stranded nucleic acid, a second homologous sequence homologous to the second double-stranded nucleic acid, and a 3' flanking adaptor sequence, wherein the first, second, and third double-stranded nucleic acids comprise non-homologous sequences at end regions; and

(d) mixing the first, second, and third double-stranded nucleic acids with a reaction mixture comprising an exonuclease, an endonuclease, a polymerase, and a ligase.

14. The method of claim 13, wherein the exonuclease is exonuclease III.

15. The method of claim 13, wherein the endonuclease is a flap endonuclease.

16. The method of claim 15, wherein the flap endonuclease is flap endonuclease 1, exonuclease 1, XPG, Dna2, or GEN 1.

17. The method of claim 13, wherein the polymerase has 5 'to 3' polymerase activity.

18. The method of claim 13, wherein the polymerase is a DNA polymerase.

19. The method of claim 13, wherein the ligase catalyzes the ligation of at least two nucleic acids.

20. The method of claim 13, wherein the homologous sequence of the first double-stranded nucleic acid or the homologous sequence of the second double-stranded nucleic acid is about 10 to about 100 base pairs.

21. The method of claim 16, wherein flap endonuclease 1 is provided at a concentration of about 0.32U to about 4.8U.

22. The method of claim 16, wherein flap endonuclease 1 is provided at a concentration of less than about 5.0U.

23. The method of claim 14, wherein the concentration of exonuclease III is in the range of about 0.1U to about 10U.

24. The method of claim 17, wherein the concentration of the polymerase is in the range of about 0.01U to about 2U.

25. The method of claim 19, wherein the concentration of the ligase is at most about 2.0U.

26. The method of claim 19, wherein the concentration of the ligase is in a range from about 4.0U to about 8.0U.

27. The method of claim 13, wherein the first double-stranded nucleic acid, the second double-stranded nucleic acid, or the third double-stranded nucleic acid, or any combination thereof, is a linear fragment.

28. The method of claim 13, wherein the product after step (d) is a linear fragment.

29. The method of claim 13, wherein the product after step (d) is a circular fragment.

30. A method of nucleic acid assembly comprising:

(a) providing a first double-stranded nucleic acid comprising, in 5 'to 3' order: a 5 'flanking adaptor sequence, a homologous sequence, an insertion sequence and a 3' flanking adaptor sequence;

(b) providing a second double stranded nucleic acid comprising, in 5 'to 3' order: a 5 'flanking adaptor sequence, a homologous sequence, an insertion sequence and a 3' flanking adaptor sequence; and

(c) mixing the first and second double-stranded nucleic acids with a reaction mixture comprising an exonuclease, an endonuclease, a polymerase, and a ligase.

31. The method of claim 30, wherein the exonuclease is exonuclease III.

32. The method of claim 30, wherein the endonuclease is a flap endonuclease.

33. The method of claim 32, wherein the flap endonuclease is flap endonuclease 1, exonuclease 1, XPG, Dna2, or GEN 1.

34. The method of claim 30, wherein the polymerase has 5 'to 3' polymerase activity.

35. The method of claim 30, wherein the polymerase is a DNA polymerase.

36. The method of claim 30, wherein the ligase catalyzes the ligation of at least two nucleic acids.

37. The method of claim 30, wherein the homologous sequence of the first double-stranded nucleic acid or the homologous sequence of the second double-stranded nucleic acid is about 10 to about 100 base pairs.

38. The method of claim 33, wherein flap endonuclease 1 is provided at a concentration of about 0.32U to about 4.8U.

39. The method of claim 33, wherein flap endonuclease 1 is provided at a concentration of less than about 5.0U.

40. The method of claim 31, wherein the concentration of exonuclease III is in the range of about 0.1U to about 10U.

41. The method of claim 34, wherein the concentration of the polymerase is in the range of about 0.1U to about 2U.

42. The method of claim 34, wherein the concentration of the polymerase is in the range of about 0.01U to about 0.2U.

43. The method of claim 36, wherein the concentration of the ligase is at most about 2.0U.

44. The method of claim 36, wherein the concentration of the ligase is in the range of about 4.0U to about 8.0U.

45. The method of claim 30, wherein the first double-stranded nucleic acid or the second double-stranded nucleic acid, or any combination thereof, is a linear fragment.

46. The method of claim 30, wherein the product after step (c) is a linear fragment.

47. The method of claim 30, wherein the product after step (c) is a circular fragment.

48. A method of nucleic acid assembly comprising:

(a) providing a first double-stranded nucleic acid comprising, in 5 'to 3' order: a 5 'flanking adaptor sequence, a homologous sequence, an insertion sequence and a 3' flanking adaptor sequence;

(b) providing a second double stranded nucleic acid comprising, in 5 'to 3' order: a 5 'flanking adaptor sequence, a homologous sequence, an insertion sequence and a 3' flanking adaptor sequence; and

(c) mixing the first and second double-stranded nucleic acids with a reaction mixture comprising an exonuclease, an endonuclease, a polymerase, and a ligase at a temperature of about 30 ℃ to about 60 ℃.

49. The method of claim 48, wherein the exonuclease is exonuclease III.

50. The method of claim 48, wherein the endonuclease is a flap endonuclease.

51. The method of claim 50, wherein the flap endonuclease is flap endonuclease 1, exonuclease 1, XPG, Dna2, or GEN 1.

52. The method of claim 48, wherein the polymerase has 5 'to 3' polymerase activity.

53. The method of claim 48, wherein the polymerase is a DNA polymerase.

54. The method of claim 48, wherein the ligase catalyzes the ligation of at least two nucleic acids.

55. The method of claim 48, wherein the homologous sequence of the first double-stranded nucleic acid or the homologous sequence of the second double-stranded nucleic acid is about 10 to about 100 base pairs.

56. The method of claim 51, wherein the concentration of flap endonuclease 1 is in the range of about 0.32U to about 4.8U.

57. The method of claim 49, wherein the concentration of exonuclease III is in the range of about 0.1U to about 10U.

58. The method of claim 52, wherein the concentration of the polymerase is in the range of about 0.01U to about 2U.

59. The method of claim 54, wherein the concentration of the ligase is at most about 2.0U.

60. The method of claim 54, wherein the concentration of the ligase is in the range of about 4.0U to about 8.0U.

61. The method of claim 48, wherein said first double-stranded nucleic acid or said second double-stranded nucleic acid, or any combination thereof, is a linear fragment.

62. The method of claim 48, wherein the product after step (c) is a linear fragment.

63. The method of claim 48, wherein the product after step (c) is a circular fragment.

64. A method of nucleic acid assembly comprising:

(a) providing a first double-stranded nucleic acid comprising, in 5 'to 3' order: 5 'flanking adaptor, homologous sequence, insertion sequence and 3' flanking adaptor sequence;

(b) providing a second double stranded nucleic acid comprising, in 5 'to 3' order: 5 'flanking adaptor, homologous sequence, insertion sequence and 3' flanking adaptor sequence; and

(c) mixing the first and second double-stranded nucleic acids with a reaction mixture comprising an endonuclease, a polymerase, and a ligase, wherein the endonuclease results in a 5' overhang.

65. A method of nucleic acid assembly comprising:

(a) providing a first double-stranded nucleic acid comprising, in 5 'to 3' order: a 5 'flanking adaptor sequence, a homologous sequence, an insertion sequence and a 3' flanking adaptor sequence;

(b) providing a second double stranded nucleic acid comprising, in 5 'to 3' order: a 5 'flanking adaptor sequence, a homologous sequence, an insertion sequence and a 3' flanking adaptor sequence; and

(c) mixing the first and second double-stranded nucleic acids with a reaction mixture comprising about 0.32U to about 4.8U endonuclease, about 0.01U to about 2U polymerase, and up to about 2.0U ligase.

66. The method of claim 65, wherein the reaction mixture further comprises about 0.5U to about 1.0U of an exonuclease.

67. A method of nucleic acid assembly comprising:

(a) providing a first double-stranded nucleic acid comprising, in 5 'to 3' order: a 5 'flanking adaptor sequence, a homologous sequence, an insertion sequence and a 3' flanking adaptor sequence;

(b) providing a second double stranded nucleic acid comprising, in 5 'to 3' order: a 5 'flanking adaptor sequence, a homologous sequence, an insertion sequence and a 3' flanking adaptor sequence; and

(c) mixing the first double stranded nucleic acid and the second double stranded nucleic acid with a reaction mixture comprising at least one enzyme having 3 'or 5' exonuclease activity, a polymerase, and a ligase, wherein the at least one enzyme having 3 'or 5' exonuclease activity removes either the 5 'flanking adaptor sequence or the 3' flanking adaptor sequence.

68. The method of claim 67, wherein the at least one enzyme having 3 'or 5' exonuclease activity is exonuclease III.

69. The method of claim 67, wherein the polymerase has 5 'to 3' polymerase activity.

70. The method of claim 67, wherein the polymerase is a DNA polymerase.

71. The method of claim 67, wherein the ligase catalyzes the ligation of at least two nucleic acids.

72. The method of claim 67, wherein the homologous sequence of the first double-stranded nucleic acid or the homologous sequence of the second double-stranded nucleic acid is about 40 base pairs.

73. The method of claim 67, wherein the homologous sequence of the first double-stranded nucleic acid or the homologous sequence of the second double-stranded nucleic acid is about 10 to about 100 base pairs.

74. The method of claim 67, wherein the homologous sequence of the first double-stranded nucleic acid or the homologous sequence of the second double-stranded nucleic acid is about 20 to about 80 base pairs.

75. The method of claim 67, wherein the concentration of exonuclease III is in the range of from about 0.1U to about 10U.

76. A method of nucleic acid assembly comprising:

(a) providing at least 10 different fragments, wherein each of the at least 10 different fragments does not comprise a terminal region having sequence homology to another of the at least 10 different fragments; and

(b) mixing the at least 10 different fragments with a plurality of enzymes, wherein the plurality of enzymes is selected from the group consisting of endonucleases, exonucleases, polymerases, and ligases, to form nucleic acids.

77. A method of nucleic acid assembly comprising:

(a) providing a plurality of polynucleotides, wherein each of said polynucleotides does not comprise terminal regions having sequence homology to another polynucleotide of said plurality; and

(b) mixing the plurality of polynucleotides with a 3 'to 5' exonuclease, a thermostable endonuclease, a high fidelity polymerase, and a thermostable ligase, wherein the plurality of polynucleotides anneal in a continuous predetermined order based on complementary sequences between adjacent polynucleotides.

Background

De novo nucleic acid synthesis is a powerful tool for basic biological research and biotechnological applications. Although various methods are known for synthesizing relatively short fragments of nucleic acids on a small scale, these techniques are unsatisfactory in terms of scalability, automation, speed, accuracy, and cost. Thus, there remains a need for efficient seamless nucleic acid assembly methods.

Disclosure of Invention

Provided herein are methods of nucleic acid synthesis and assembly, comprising: (a) providing a plurality of polynucleotides, and (b) mixing the plurality of polynucleotides with an exonuclease, a flap endonuclease, a polymerase, and a ligase, wherein the plurality of polynucleotides anneal in a continuous predetermined order based on complementary sequences between adjacent polynucleotides. Further provided herein are nucleic acid synthesis and assembly methods, wherein the exonuclease is exonuclease III. Further provided herein are nucleic acid synthesis and assembly methods, wherein the flap endonuclease is flap endonuclease 1, exonuclease 1, XPG, Dna2, or GEN 1. Further provided herein are nucleic acid synthesis and assembly methods, wherein the polymerase has 5 'to 3' polymerase activity. Further provided herein are nucleic acid synthesis and assembly methods, wherein the polymerase is a DNA polymerase. Further provided herein are nucleic acid synthesis and assembly methods, wherein the ligase catalyzes the ligation of at least two nucleic acids. Further provided herein are nucleic acid synthesis and assembly methods, wherein the concentration of flap endonuclease 1 is in the range of about 0.32U to about 4.8U. Further provided herein are nucleic acid synthesis and assembly methods, wherein the concentration of exonuclease III is in the range of about 0.1U to about 10U. Further provided herein are nucleic acid synthesis and assembly methods, wherein the concentration of exonuclease III is in the range of about 0.5U to about 1.0U. Further provided herein are nucleic acid synthesis and assembly methods, wherein the concentration of exonuclease III is in the range of about 1.0U to about 2.0U. Further provided herein are nucleic acid synthesis and assembly methods, wherein the concentration of the polymerase is in the range of about 0.1U to about 2U. Further provided herein are nucleic acid synthesis and assembly methods, wherein the polymerase is at a concentration of about 0.1U. Further provided herein are nucleic acid synthesis and assembly methods, wherein the polymerase is at a concentration of about 0.2U. Further provided herein are nucleic acid synthesis and assembly methods, wherein the ligase is at a concentration of up to about 2.0U. Further provided herein are nucleic acid synthesis and assembly methods, wherein the concentration of the ligase is in the range of about 4.0U to about 8.0U.

Provided herein are methods of nucleic acid synthesis and assembly, comprising: (a) providing a first double-stranded nucleic acid; (b) providing a second double-stranded nucleic acid; (c) providing a third double stranded nucleic acid comprising, in 5 'to 3' order: a 5 'flanking adaptor sequence, a first homologous sequence homologous to the first double-stranded nucleic acid, a second homologous sequence homologous to the second double-stranded nucleic acid, and a 3' flanking adaptor sequence; and (d) mixing the first, second, and third double-stranded nucleic acids with a reaction mixture comprising an exonuclease, a flap endonuclease, a polymerase, and a ligase. Further provided herein are nucleic acid synthesis and assembly methods, wherein the exonuclease is exonuclease III. Further provided herein are nucleic acid synthesis and assembly methods, wherein the flap endonuclease is flap endonuclease 1, exonuclease 1, XPG, Dna2, or GEN 1. Further provided herein are nucleic acid synthesis and assembly methods, wherein the amount of flap endonuclease 1 provided is from about 0.32U to about 4.8U. Further provided herein are nucleic acid synthesis and assembly methods, wherein the amount of flap endonuclease 1 provided is less than about 5.0U. Further provided herein are nucleic acid synthesis and assembly methods, wherein the polymerase has 5 'to 3' polymerase activity. Further provided herein are nucleic acid synthesis and assembly methods, wherein the polymerase is a DNA polymerase. Further provided herein are nucleic acid synthesis and assembly methods, wherein the ligase catalyzes the ligation of at least two nucleic acids. Further provided herein are nucleic acid synthesis and assembly methods, wherein the first homologous sequence or the second homologous sequence is about 10 to about 100 base pairs. Further provided herein are nucleic acid synthesis and assembly methods, wherein the first homologous sequence and the second homologous sequence are each independently about 10 to about 100 base pairs. Further provided herein are nucleic acid synthesis and assembly methods, wherein the first homologous sequence or the second homologous sequence is about 20 to about 80 base pairs. Further provided herein are nucleic acid synthesis and assembly methods, wherein the first homologous sequence and the second homologous sequence are each independently about 20 to about 80 base pairs. Further provided herein are methods of nucleic acid synthesis and assembly, wherein the first homologous sequence or the second homologous sequence is about 40 base pairs. Further provided herein are methods of nucleic acid synthesis and assembly, wherein the first homologous sequence and the second homologous sequence are each independently about 40 base pairs. Further provided herein are nucleic acid synthesis and assembly methods, wherein the concentration of exonuclease III is in the range of about 0.1U to about 10U. Further provided herein are nucleic acid synthesis and assembly methods, wherein the concentration of exonuclease III is in the range of about 0.5U to about 1.0U. Further provided herein are nucleic acid synthesis and assembly methods, wherein the concentration of exonuclease III is in the range of about 1.0U to about 2.0U. Further provided herein are nucleic acid synthesis and assembly methods, wherein the concentration of the polymerase is present in an amount from about 0.1U to about 2U. Further provided herein are nucleic acid synthesis and assembly methods, wherein the polymerase is at a concentration of about 0.1U to about 0.2U. Further provided herein are nucleic acid synthesis and assembly methods, wherein the ligase is at a concentration of up to about 2.0U. Further provided herein are nucleic acid synthesis and assembly methods, wherein the concentration of the ligase is in the range of about 4.0U to about 8.0U. Further provided herein are nucleic acid synthesis and assembly methods, wherein the concentration of the ligase is in the range of about 0.5U to about 1.0U. Further provided herein are nucleic acid synthesis and assembly methods, wherein the first, second, or third double-stranded nucleic acid, or any combination thereof, is a linear fragment. Further provided herein are nucleic acid synthesis and assembly methods, wherein the product after step (d) is a linear fragment. Further provided herein are nucleic acid synthesis and assembly methods, wherein the product after step (d) is a circular fragment.

Provided herein are methods of nucleic acid assembly comprising: (a) providing a first double-stranded nucleic acid comprising, in 5 'to 3' order: a 5 'flanking adaptor sequence, a homologous sequence, an insertion sequence and a 3' flanking adaptor sequence; (b) providing a second double stranded nucleic acid comprising, in 5 'to 3' order: a 5 'flanking adaptor sequence, a homologous sequence, an insertion sequence and a 3' flanking adaptor sequence; and (c) mixing the first and second double-stranded nucleic acids with a reaction mixture comprising an exonuclease, a flap endonuclease, a polymerase, and a ligase. Further provided herein are nucleic acid assembly methods, wherein the exonuclease is exonuclease III. Further provided herein are nucleic acid assembly methods, wherein the flap endonuclease is flap endonuclease 1, exonuclease 1, XPG, Dna2, or GEN 1. Further provided herein are nucleic acid assembly methods, wherein the amount of flap endonuclease 1 provided is about 0.32U to about 4.8U. Further provided herein are nucleic acid assembly methods, wherein the amount of flap endonuclease 1 provided is less than about 5.0U. Further provided herein are nucleic acid assembly methods, wherein the polymerase has 5 'to 3' polymerase activity. Further provided herein are nucleic acid assembly methods, wherein the polymerase is a DNA polymerase. Further provided herein are nucleic acid assembly methods, wherein the ligase catalyzes the ligation of at least two nucleic acids. Further provided herein are nucleic acid assembly methods, wherein the homologous sequence of the first double-stranded nucleic acid or the homologous sequence of the second double-stranded nucleic acid is about 10 to about 100 base pairs. Further provided herein are nucleic acid assembly methods, wherein the homologous sequence of the first double-stranded nucleic acid and the homologous sequence of the second double-stranded nucleic acid are each independently about 10 to about 100 base pairs. Further provided herein are nucleic acid assembly methods, wherein the homologous sequence of the first double-stranded nucleic acid or the homologous sequence of the second double-stranded nucleic acid is about 20 to about 80 base pairs. Further provided herein are nucleic acid assembly methods, wherein the homologous sequence of the first double-stranded nucleic acid and the homologous sequence of the second double-stranded nucleic acid are each independently about 20 to about 80 base pairs. Further provided herein are methods of nucleic acid assembly, wherein the homologous sequence of the first double-stranded nucleic acid or the homologous sequence of the second double-stranded nucleic acid is about 40 base pairs. Further provided herein are methods of nucleic acid assembly, wherein the homologous sequence of the first double-stranded nucleic acid and the homologous sequence of the second double-stranded nucleic acid are each independently about 40 base pairs. Further provided herein are nucleic acid assembly methods, wherein the concentration of exonuclease III is in the range of about 0.1U to about 10U. Further provided herein are nucleic acid assembly methods, wherein the concentration of exonuclease III is in the range of about 0.5U to about 1.0U. Further provided herein are nucleic acid assembly methods, wherein the concentration of exonuclease III is in the range of about 1.0U to about 2.0U. Further provided herein are nucleic acid assembly methods, wherein the concentration of the polymerase is present in an amount from about 0.1U to about 2U. Further provided herein are nucleic acid assembly methods, wherein the concentration of the polymerase is from about 0.1U to about 0.2U. Further provided herein are nucleic acid assembly methods, wherein the concentration of the ligase is at most about 2.0U. Further provided herein are nucleic acid assembly methods, wherein the concentration of the ligase is in a range from about 4.0U to about 8.0U. Further provided herein are nucleic acid assembly methods, wherein the concentration of the ligase is in the range of about 0.5U to about 1.0U. Further provided herein are nucleic acid assembly methods, wherein the first double-stranded nucleic acid or the second double-stranded nucleic acid is a linear fragment. Further provided herein are nucleic acid assembly methods, wherein the product after step (c) is a linear fragment. Further provided herein are nucleic acid assembly methods, wherein the product after step (c) is a circular fragment.

Provided herein are methods of nucleic acid assembly comprising: (a) providing a first double-stranded nucleic acid comprising, in 5 'to 3' order: a 5 'flanking adaptor sequence, a homologous sequence, an insertion sequence and a 3' flanking adaptor sequence; (b) providing a second double stranded nucleic acid comprising, in 5 'to 3' order: a 5 'flanking adaptor sequence, a homologous sequence, an insertion sequence and a 3' flanking adaptor sequence; and (c) mixing the first and second double-stranded nucleic acids with a reaction mixture comprising an exonuclease, a flap endonuclease, a polymerase, and a ligase at a temperature of about 30 ℃ to about 60 ℃. Further provided herein are nucleic acid assembly methods, wherein the exonuclease is exonuclease III. Further provided herein are nucleic acid assembly methods, wherein the flap endonuclease is flap endonuclease 1, exonuclease 1, XPG, Dna2, or GEN 1. Further provided herein are nucleic acid assembly methods, wherein the concentration of flap endonuclease 1 is in the range of about 0.32U to about 4.8U. Further provided herein are nucleic acid assembly methods, wherein the polymerase has 5 'to 3' polymerase activity. Further provided herein are nucleic acid assembly methods, wherein the polymerase is a DNA polymerase. Further provided herein are nucleic acid assembly methods, wherein the ligase catalyzes the ligation of at least two nucleic acids. Further provided herein are nucleic acid assembly methods, wherein the homologous sequence of the first double-stranded nucleic acid or the homologous sequence of the second double-stranded nucleic acid is about 10 to about 100 base pairs. Further provided herein are nucleic acid assembly methods, wherein the homologous sequence of the first double-stranded nucleic acid and the homologous sequence of the second double-stranded nucleic acid are each independently about 10 to about 100 base pairs. Further provided herein are nucleic acid assembly methods, wherein the homologous sequence of the first double-stranded nucleic acid or the homologous sequence of the second double-stranded nucleic acid is about 20 to about 80 base pairs. Further provided herein are nucleic acid assembly methods, wherein the homologous sequence of the first double-stranded nucleic acid and the homologous sequence of the second double-stranded nucleic acid are each independently about 20 to about 80 base pairs. Further provided herein are methods of nucleic acid assembly, wherein the homologous sequence of the first double-stranded nucleic acid or the homologous sequence of the second double-stranded nucleic acid is about 40 base pairs. Further provided herein are methods of nucleic acid assembly, wherein the homologous sequence of the first double-stranded nucleic acid and the homologous sequence of the second double-stranded nucleic acid are each independently about 40 base pairs. Further provided herein are nucleic acid assembly methods, wherein the concentration of exonuclease III is in the range of about 0.1U to about 10U. Further provided herein are nucleic acid assembly methods, wherein the concentration of exonuclease III is in the range of about 0.5U to about 1.0U. Further provided herein are nucleic acid assembly methods, wherein the concentration of exonuclease III is in the range of about 1.0U to about 2.0U. Further provided herein are nucleic acid assembly methods, wherein the concentration of the polymerase is in the range of about 0.1U to about 2U. Further provided herein are nucleic acid assembly methods, wherein the polymerase is at a concentration of about 0.1U. Further provided herein are nucleic acid assembly methods, wherein the polymerase is at a concentration of about 0.2U. Further provided herein are nucleic acid assembly methods, wherein the concentration of the ligase is at most about 2.0U. Further provided herein are nucleic acid assembly methods, wherein the concentration of the ligase is in a range from about 4.0U to about 8.0U. Further provided herein are nucleic acid assembly methods, wherein the concentration of the ligase is in the range of 0.5U to about 1.0U. Further provided herein are nucleic acid assembly methods, wherein the first double-stranded nucleic acid or the second double-stranded nucleic acid is a linear fragment. Further provided herein are nucleic acid assembly methods, wherein the product after step (c) is a linear fragment. Further provided herein are nucleic acid assembly methods, wherein the product after step (c) is a circular fragment.

Provided herein are methods of nucleic acid assembly comprising: (a) providing a first double-stranded nucleic acid comprising, in 5 'to 3' order: 5 'flanking adaptor, homologous sequence, insertion sequence and 3' flanking adaptor sequence; (b) providing a second double stranded nucleic acid comprising, in 5 'to 3' order: 5 'flanking adaptor, homologous sequence, insertion sequence and 3' flanking adaptor sequence; and (c) mixing the first and second double-stranded nucleic acids with a reaction mixture comprising a flap endonuclease, a polymerase, and a ligase, wherein the flap endonuclease results in a 5' overhang.

Provided herein are methods of nucleic acid assembly comprising: (a) providing a first double-stranded nucleic acid comprising, in 5 'to 3' order: a 5 'flanking adaptor sequence, a homologous sequence, an insertion sequence and a 3' flanking adaptor sequence; (b) providing a second double stranded nucleic acid comprising, in 5 'to 3' order: a 5 'flanking adaptor sequence, a homologous sequence, an insertion sequence and a 3' flanking adaptor sequence; and (c) mixing the first and second double-stranded nucleic acids with a reaction mixture comprising about 0.5U to about 1.0U exonuclease, about 0.32U to about 4.8U flap endonuclease, about 0.1U to about 2U polymerase, and up to about 2.0U ligase.

Provided herein are methods of nucleic acid assembly comprising: (a) providing a first double-stranded nucleic acid comprising, in 5 'to 3' order: a 5 'flanking adaptor sequence, a homologous sequence, an insertion sequence and a 3' flanking adaptor sequence; (b) providing a second double stranded nucleic acid comprising, in 5 'to 3' order: a 5 'flanking adaptor sequence, a homologous sequence, an insertion sequence and a 3' flanking adaptor sequence; and (c) mixing the first and second double-stranded nucleic acids with a reaction mixture comprising about 0.32U to about 4.8U flap endonuclease, about 0.1U to about 2U polymerase, and up to about 2.0U ligase.

Provided herein are methods of nucleic acid assembly comprising: (a) providing a plurality of polynucleotides, wherein each of said polynucleotides does not comprise terminal regions having sequence homology to another polynucleotide of said plurality; and (b) mixing the plurality of polynucleotides with an exonuclease, an endonuclease, a polymerase, and a ligase, wherein the plurality of polynucleotides anneal in a continuous predetermined order based on the complementary sequence between adjacent polynucleotides. Further provided herein are nucleic acid assembly methods, wherein the exonuclease is exonuclease III. Further provided herein are nucleic acid assembly methods, wherein the endonuclease is a flap endonuclease. Further provided herein are nucleic acid assembly methods, wherein the flap endonuclease is flap endonuclease 1, exonuclease 1, XPG, Dna2, or GEN 1. Further provided herein are nucleic acid assembly methods, wherein the concentration of flap endonuclease 1 is in the range of about 0.32U to about 4.8U. Further provided herein are nucleic acid assembly methods, wherein the polymerase has 5 'to 3' polymerase activity. Further provided herein are nucleic acid assembly methods, wherein the polymerase is a DNA polymerase. Further provided herein are nucleic acid assembly methods, wherein the ligase catalyzes the ligation of at least two nucleic acids. Further provided herein are nucleic acid assembly methods, wherein the concentration of exonuclease III is in the range of about 0.1U to about 10U. Further provided herein are nucleic acid assembly methods, wherein the concentration of the polymerase is in the range of about 0.01U to about 2U. Further provided herein are nucleic acid assembly methods, wherein the polymerase is at a concentration of about 0.1U. Further provided herein are nucleic acid assembly methods, wherein the polymerase is at a concentration of about 0.01U. Further provided herein are nucleic acid assembly methods, wherein the concentration of the ligase is at most about 2.0U.

Provided herein are methods of nucleic acid assembly comprising: (a) providing a first double-stranded nucleic acid; (b) providing a second double-stranded nucleic acid; (c) providing a third double stranded nucleic acid comprising, in 5 'to 3' order: a 5 'flanking adaptor sequence, a first homologous sequence homologous to the first double-stranded nucleic acid, a second homologous sequence homologous to the second double-stranded nucleic acid, and a 3' flanking adaptor sequence, wherein the first, second, and third double-stranded nucleic acids comprise non-homologous sequences at end regions; and (d) mixing the first, second, and third double-stranded nucleic acids with a reaction mixture comprising an exonuclease, an endonuclease, a polymerase, and a ligase. Further provided herein are nucleic acid assembly methods, wherein the exonuclease is exonuclease III. Further provided herein are nucleic acid assembly methods, wherein the endonuclease is a flap endonuclease. Further provided herein are nucleic acid assembly methods, wherein the flap endonuclease is flap endonuclease 1, exonuclease 1, XPG, Dna2, or GEN 1. Further provided herein are nucleic acid assembly methods, wherein flap endonuclease 1 is provided at a concentration of about 0.32U to about 4.8U. Further provided herein are nucleic acid assembly methods, wherein the endonuclease is flap endonuclease 1 provided at a concentration of less than about 5.0U. Further provided herein are nucleic acid assembly methods, wherein the polymerase has 5 'to 3' polymerase activity. Further provided herein are nucleic acid assembly methods, wherein the polymerase is a DNA polymerase. Further provided herein are nucleic acid assembly methods, wherein the ligase catalyzes the ligation of at least two nucleic acids. Further provided herein are nucleic acid assembly methods, wherein the homologous sequence of the first double-stranded nucleic acid or the homologous sequence of the second double-stranded nucleic acid is about 10 to about 100 base pairs. Further provided herein are nucleic acid assembly methods, wherein the homologous sequence of the first double-stranded nucleic acid and the homologous sequence of the second double-stranded nucleic acid are each independently about 10 to about 100 base pairs. Further provided herein are nucleic acid assembly methods, wherein the homologous sequence of the first double-stranded nucleic acid or the homologous sequence of the second double-stranded nucleic acid is about 20 to about 80 base pairs. Further provided herein are nucleic acid assembly methods, wherein the homologous sequence of the first double-stranded nucleic acid and the homologous sequence of the second double-stranded nucleic acid are each independently about 20 to about 80 base pairs. Further provided herein are methods of nucleic acid assembly, wherein the homologous sequence of the first double-stranded nucleic acid or the homologous sequence of the second double-stranded nucleic acid is about 40 base pairs. Further provided herein are methods of nucleic acid assembly, wherein the homologous sequence of the first double-stranded nucleic acid and the homologous sequence of the second double-stranded nucleic acid are each independently about 40 base pairs. Further provided herein are nucleic acid assembly methods, wherein the concentration of exonuclease III is in the range of about 0.1U to about 10U. Further provided herein are nucleic acid assembly methods, wherein the concentration of the polymerase is in the range of about 0.01U to about 2U. Further provided herein are nucleic acid assembly methods, wherein the concentration of the ligase is at most about 2.0U. Further provided herein are nucleic acid assembly methods, wherein the first double-stranded nucleic acid, the second double-stranded nucleic acid, or the third double-stranded nucleic acid, or any combination thereof, is a linear fragment. Further provided herein are nucleic acid assembly methods, wherein the product after step (d) is a linear fragment. Further provided herein are nucleic acid assembly methods, wherein the product after step (d) is a circular fragment.

Provided herein are methods of nucleic acid assembly comprising: (a) providing a first double-stranded nucleic acid comprising, in 5 'to 3' order: a 5 'flanking adaptor sequence, a homologous sequence, an insertion sequence and a 3' flanking adaptor sequence; (b) providing a second double stranded nucleic acid comprising, in 5 'to 3' order: a 5 'flanking adaptor sequence, a homologous sequence, an insertion sequence and a 3' flanking adaptor sequence; and (c) mixing the first and second double-stranded nucleic acids with a reaction mixture comprising an exonuclease, an endonuclease, a polymerase, and a ligase. Further provided herein are nucleic acid assembly methods, wherein the exonuclease is exonuclease III. Further provided herein are nucleic acid assembly methods, wherein the endonuclease is a flap endonuclease. Further provided herein are nucleic acid assembly methods, wherein the flap endonuclease is flap endonuclease 1, exonuclease 1, XPG, Dna2, or GEN 1. Further provided herein are nucleic acid assembly methods, wherein flap endonuclease 1 is provided at a concentration of about 0.32U to about 4.8U. Further provided herein are nucleic acid assembly methods, wherein flap endonuclease 1 is provided at a concentration of less than about 5.0U. Further provided herein are nucleic acid assembly methods, wherein the polymerase has 5 'to 3' polymerase activity. Further provided herein are nucleic acid assembly methods, wherein the polymerase is a DNA polymerase. Further provided herein are nucleic acid assembly methods, wherein the ligase catalyzes the ligation of at least two nucleic acids. Further provided herein are nucleic acid assembly methods, wherein the homologous sequence of the first double-stranded nucleic acid or the homologous sequence of the second double-stranded nucleic acid is about 10 to about 100 base pairs. Further provided herein are nucleic acid assembly methods, wherein the homologous sequence of the first double-stranded nucleic acid and the homologous sequence of the second double-stranded nucleic acid are each independently about 10 to about 100 base pairs. Further provided herein are nucleic acid assembly methods, wherein the homologous sequence of the first double-stranded nucleic acid or the homologous sequence of the second double-stranded nucleic acid is about 20 to about 80 base pairs. Further provided herein are nucleic acid assembly methods, wherein the homologous sequence of the first double-stranded nucleic acid and the homologous sequence of the second double-stranded nucleic acid are each independently about 20 to about 80 base pairs. Further provided herein are methods of nucleic acid assembly, wherein the homologous sequence of the first double-stranded nucleic acid or the homologous sequence of the second double-stranded nucleic acid is about 40 base pairs. Further provided herein are methods of nucleic acid assembly, wherein the homologous sequence of the first double-stranded nucleic acid and the homologous sequence of the second double-stranded nucleic acid are each independently about 40 base pairs. Further provided herein are nucleic acid assembly methods, wherein the concentration of exonuclease III is in the range of about 0.1U to about 10U. Further provided herein are nucleic acid assembly methods, wherein the concentration of the polymerase is in the range of about 0.1U to about 2U. Further provided herein are nucleic acid assembly methods, wherein the concentration of the polymerase is from about 0.01U to about 0.2U. Further provided herein are nucleic acid assembly methods, wherein the concentration of the ligase is at most about 2.0U. Further provided herein are nucleic acid assembly methods, wherein the first double-stranded nucleic acid or the second double-stranded nucleic acid, or any combination thereof, is a linear fragment. Further provided herein are nucleic acid assembly methods, wherein the product after step (c) is a linear fragment. Further provided herein are nucleic acid assembly methods, wherein the product after step (c) is a circular fragment.

Provided herein are methods of nucleic acid assembly comprising: (a) providing a first double-stranded nucleic acid comprising, in 5 'to 3' order: a 5 'flanking adaptor sequence, a homologous sequence, an insertion sequence and a 3' flanking adaptor sequence; (b) providing a second double stranded nucleic acid comprising, in 5 'to 3' order: a 5 'flanking adaptor sequence, a homologous sequence, an insertion sequence and a 3' flanking adaptor sequence; and (c) mixing the first and second double-stranded nucleic acids with a reaction mixture comprising an exonuclease, an endonuclease, a polymerase, and a ligase at a temperature of about 30 ℃ to about 60 ℃. Further provided herein are nucleic acid assembly methods, wherein the exonuclease is exonuclease III. Further provided herein are nucleic acid assembly methods, wherein the endonuclease is a flap endonuclease. Further provided herein are nucleic acid assembly methods, wherein the flap endonuclease is flap endonuclease 1, exonuclease 1, XPG, Dna2, or GEN 1. Further provided herein are nucleic acid assembly methods, wherein the concentration of flap endonuclease 1 is in the range of about 0.32U to about 4.8U. Further provided herein are nucleic acid assembly methods, wherein the polymerase has 5 'to 3' polymerase activity. Further provided herein are nucleic acid assembly methods, wherein the polymerase is a DNA polymerase. Further provided herein are nucleic acid assembly methods, wherein the ligase catalyzes the ligation of at least two nucleic acids. Further provided herein are nucleic acid assembly methods, wherein the homologous sequence of the first double-stranded nucleic acid or the homologous sequence of the second double-stranded nucleic acid is about 10 to about 100 base pairs. Further provided herein are nucleic acid assembly methods, wherein the homologous sequence of the first double-stranded nucleic acid and the homologous sequence of the second double-stranded nucleic acid are each independently about 10 to about 100 base pairs. Further provided herein are nucleic acid assembly methods, wherein the homologous sequence of the first double-stranded nucleic acid or the homologous sequence of the second double-stranded nucleic acid is about 20 to about 80 base pairs. Further provided herein are nucleic acid assembly methods, wherein the homologous sequence of the first double-stranded nucleic acid and the homologous sequence of the second double-stranded nucleic acid are each independently about 20 to about 80 base pairs. Further provided herein are methods of nucleic acid assembly, wherein the homologous sequence of the first double-stranded nucleic acid or the homologous sequence of the second double-stranded nucleic acid is about 40 base pairs. Further provided herein are methods of nucleic acid assembly, wherein the homologous sequence of the first double-stranded nucleic acid and the homologous sequence of the second double-stranded nucleic acid are each independently about 40 base pairs. Further provided herein are nucleic acid assembly methods, wherein the concentration of exonuclease III is in the range of about 0.1U to about 10U. Further provided herein are nucleic acid assembly methods, wherein the concentration of the polymerase is in the range of about 0.01U to about 2U. Further provided herein are nucleic acid assembly methods, wherein the polymerase is at a concentration of about 0.1U. Further provided herein are nucleic acid assembly methods, wherein the polymerase is at a concentration of about 0.01U. Further provided herein are nucleic acid assembly methods, wherein the concentration of the ligase is at most about 2.0U. Further provided herein are nucleic acid assembly methods, wherein the first double-stranded nucleic acid or the second double-stranded nucleic acid, or any combination thereof, is a linear fragment. Further provided herein are nucleic acid assembly methods, wherein the product after step (c) is a linear fragment. Further provided herein are nucleic acid assembly methods, wherein the product after step (c) is a circular fragment.

Provided herein are methods of nucleic acid assembly comprising: (a) providing a first double-stranded nucleic acid comprising, in 5 'to 3' order: 5 'flanking adaptor, homologous sequence, insertion sequence and 3' flanking adaptor sequence; (b) providing a second double stranded nucleic acid comprising, in 5 'to 3' order: 5 'flanking adaptor, homologous sequence, insertion sequence and 3' flanking adaptor sequence; and (c) mixing the first and second double-stranded nucleic acids with a reaction mixture comprising an endonuclease, a polymerase, and a ligase, wherein the endonuclease results in a 5' overhang.

Provided herein are methods of nucleic acid assembly comprising: (a) providing a first double-stranded nucleic acid comprising, in 5 'to 3' order: a 5 'flanking adaptor sequence, a homologous sequence, an insertion sequence and a 3' flanking adaptor sequence; (b) providing a second double stranded nucleic acid comprising, in 5 'to 3' order: a 5 'flanking adaptor sequence, a homologous sequence, an insertion sequence and a 3' flanking adaptor sequence; and (c) mixing the first and second double-stranded nucleic acids with a reaction mixture comprising about 0.5U to about 1.0U exonuclease, about 0.32U to about 4.8U endonuclease, about 0.01U to about 2U polymerase, and up to about 2.0U ligase.

Provided herein are methods of nucleic acid assembly comprising: (a) providing a first double-stranded nucleic acid comprising, in 5 'to 3' order: a 5 'flanking adaptor sequence, a homologous sequence, an insertion sequence and a 3' flanking adaptor sequence; (b) providing a second double stranded nucleic acid comprising, in 5 'to 3' order: a 5 'flanking adaptor sequence, a homologous sequence, an insertion sequence and a 3' flanking adaptor sequence; and (c) mixing the first and second double-stranded nucleic acids with a reaction mixture comprising about 0.32U to about 4.8U endonuclease, about 0.01U to about 2U polymerase, and up to about 2.0U ligase.

Provided herein are methods of nucleic acid assembly comprising: (a) providing a first double-stranded nucleic acid comprising, in 5 'to 3' order: a 5 'flanking adaptor sequence, a homologous sequence, an insertion sequence and a 3' flanking adaptor sequence; (b) providing a second double stranded nucleic acid comprising, in 5 'to 3' order: a 5 'flanking adaptor sequence, a homologous sequence, an insertion sequence and a 3' flanking adaptor sequence; and (c) mixing the first double-stranded nucleic acid and the second double-stranded nucleic acid with a reaction mixture comprising at least one enzyme having 3 'or 5' exonuclease activity, a polymerase, and a ligase, wherein the at least one enzyme having 3 'or 5' exonuclease activity removes either the 5 'flanking adaptor sequence or the 3' flanking adaptor sequence. Further provided herein are nucleic acid assembly methods, wherein the at least one enzyme having 3 'or 5' exonuclease activity is exonuclease III. Further provided herein are nucleic acid assembly methods, wherein the polymerase has 5 'to 3' polymerase activity. Further provided herein are nucleic acid assembly methods, wherein the polymerase is a DNA polymerase. Further provided herein are nucleic acid assembly methods, wherein the ligase catalyzes the ligation of at least two nucleic acids. Further provided herein are methods of nucleic acid assembly, wherein the homologous sequence of the first double-stranded nucleic acid or the homologous sequence of the second double-stranded nucleic acid is about 40 base pairs. Further provided herein are methods of nucleic acid assembly, wherein the homologous sequence of the first double-stranded nucleic acid and the homologous sequence of the second double-stranded nucleic acid are each independently about 40 base pairs. Further provided herein are nucleic acid assembly methods, wherein the homologous sequence of the first double-stranded nucleic acid or the homologous sequence of the second double-stranded nucleic acid is about 10 to about 100 base pairs. Further provided herein are nucleic acid assembly methods, wherein the homologous sequence of the first double-stranded nucleic acid and the homologous sequence of the second double-stranded nucleic acid are each independently about 10 to about 100 base pairs. Further provided herein are nucleic acid assembly methods, wherein the homologous sequence of the first double-stranded nucleic acid or the homologous sequence of the second double-stranded nucleic acid is about 20 to about 80 base pairs. Further provided herein are nucleic acid assembly methods, wherein the homologous sequence of the first double-stranded nucleic acid and the homologous sequence of the second double-stranded nucleic acid are each independently about 20 to about 80 base pairs. Further provided herein are nucleic acid assembly methods, wherein the concentration of exonuclease III is in the range of about 0.1U to about 10U.

Provided herein are methods of nucleic acid assembly comprising: (a) providing at least 10 different fragments, wherein each of the at least 10 different fragments does not comprise a terminal region having sequence homology to another of the at least 10 different fragments; and (b) mixing the at least 10 different fragments with a plurality of enzymes, wherein the plurality of enzymes is selected from the group consisting of endonucleases, exonucleases, polymerases, and ligases, to form nucleic acids. Further provided herein are methods of nucleic acid assembly, wherein the nucleic acid is linked to a vector sequence. Further provided herein are methods of nucleic acid assembly, wherein the nucleic acid is 50 bases to 200 bases in length. Further provided herein are methods of nucleic acid assembly, wherein the nucleic acid is 100 bases to 2000 bases in length. Further provided herein are nucleic acid assembly methods, wherein the exonuclease is exonuclease III. Further provided herein are nucleic acid assembly methods, wherein the endonuclease is a flap endonuclease. Further provided herein are nucleic acid assembly methods, wherein the flap endonuclease is flap endonuclease 1, exonuclease 1, XPG, Dna2, or GEN 1. Further provided herein are nucleic acid assembly methods, wherein the polymerase has 5 'to 3' polymerase activity. Further provided herein are nucleic acid assembly methods, wherein the polymerase is a DNA polymerase. Further provided herein are nucleic acid assembly methods, wherein the ligase catalyzes the ligation of at least two nucleic acids.

Provided herein are methods of nucleic acid assembly comprising: (a) providing a plurality of polynucleotides, wherein each of said polynucleotides does not comprise terminal regions having sequence homology to another polynucleotide of said plurality; and (b) mixing the plurality of polynucleotides with a 3 'to 5' exonuclease, a thermostable endonuclease, a high fidelity polymerase, and a thermostable ligase, wherein the plurality of polynucleotides anneal in a continuous predetermined order based on complementary sequences between adjacent polynucleotides. Further provided herein are nucleic acid assembly methods, wherein the exonuclease is exonuclease III. Further provided herein are nucleic acid assembly methods, wherein the endonuclease is a flap endonuclease. Further provided herein are nucleic acid assembly methods, wherein the flap endonuclease is flap endonuclease 1, exonuclease 1, XPG, Dna2, or GEN 1. Further provided herein are nucleic acid assembly methods, wherein the endonuclease is flap endonuclease 1 provided at a concentration in the range of about 0.32U to about 4.8U. Further provided herein are nucleic acid assembly methods, wherein the polymerase has 5 'to 3' polymerase activity. Further provided herein are nucleic acid assembly methods, wherein the polymerase is a DNA polymerase. Further provided herein are nucleic acid assembly methods, wherein the ligase catalyzes the ligation of at least two nucleic acids. Further provided herein are nucleic acid assembly methods, wherein the concentration of exonuclease III is in the range of about 0.1U to about 10U. Further provided herein are nucleic acid assembly methods, wherein the concentration of the polymerase is in the range of about 0.01U to about 2U. Further provided herein are nucleic acid assembly methods, wherein the concentration of the ligase is at most about 2.0U.

Is incorporated by reference

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Drawings

This patent or application document contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the office upon request and payment of the necessary fee.

Figure 1A depicts a schematic of flap endonuclease-mediated nucleic acid assembly.

FIG. 1B depicts a schematic of flap endonuclease-mediated nucleic acid assembly using bridge assembly.

Figure 2 depicts a system for polynucleotide synthesis and flap endonuclease-mediated nucleic acid assembly.

Fig. 3 shows a computer system.

FIG. 4 is a block diagram illustrating an architecture of a computer system.

FIG. 5 is a block diagram of a multiprocessor computer system using a shared virtual address memory space.

FIG. 6 is a diagram illustrating a network configured to incorporate multiple computer systems, multiple cellular telephones and personal data assistants, and Network Attached Storage (NAS).

FIG. 7 is a graph from BioAnalyzer reads, in nucleotide bases on the x-axis and fluorescence units on the y-axis.

FIG. 8 is a graph from BioAnalyzer reads, in nucleotide bases on the x-axis and fluorescence units on the y-axis.

FIG. 9 is a graph of Colony Forming Units (CFU) using different concentrations of ExoIII and Fen 1.

FIG. 10 is a graph of the percent correct assembly of Colony Forming Units (CFU) and flap endonuclease-mediated nucleic acid assembly reactions using different enzyme concentrations.

FIG. 11 is a diagram of Colony Forming Units (CFU) of the 1.8kb assembly.

FIG. 12 is a diagram of flap endonuclease-mediated nucleic acid assembly using two DNA fragments.

FIG. 13 is a graph of colony forming units (y-axis) for several genes (x-axis) when multiple DNA fragments are assembled into a DNA vector using flap endonuclease-mediated nucleic acid assembly.

Fig. 14A is a graph of colony forming units (y-axis) for several genes (x-axis) when multiple DNA fragments were assembled into DNA vectors by flap endonuclease-mediated nucleic acid assembly with increasing concentrations of ExoIII.

FIG. 14B is a diagram of the next generation sequence analysis of the assembled genes.

Fig. 14C is a graph of a sample of the assembly rate.

Fig. 15A is a graph of average Colony Forming Units (CFUs) (y-axis) of flap endonuclease-mediated nucleic acid assembly using nucleic acid bridges.

Fig. 15B is a graph of log scale Colony Forming Units (CFUs) (y-axis) of flap endonuclease-mediated nucleic acid assembly using nucleic acid bridges.

FIG. 16A is a graph of colony forming units (CFU, y-axis) for nucleic acid assembly of a flap endonuclease-mediated gene (x-axis) using 1,2, 3, 4,5, 6, 7, 8, 9, 10, 11, or 12 fragments.

FIG. 16B is a graph of the next generation sequence analysis of the percentage population sequencing (y-axis) and assembled genes (x-axis) using 1,2, 3, 4,5, 6, 7, 8, 9, 10, 11, or 12 fragments.

FIG. 16C is a graph showing the passage rate (y-axis) of a gene (x-axis) when 1,2, 3, 4,5, 6, 7, 8, 9, 10, 11 or 12 fragments were used.

FIG. 17 is a graph of colony forming units (CFU, y-axis) of flap endonuclease-mediated nucleic acid assembly using method 2 and method 3 for both genes, using incubation times of 10 and 30 minutes.

FIG. 18A is a graph from BioAnalyzer reads, nucleotide bases on the x-axis and fluorescence units on the y-axis.

Fig. 18B is a graph from the BioAnalyzer reading with incubation time on the x-axis and fluorescence units on the y-axis.

FIGS. 19A-19B are graphs of colony forming units (y-axis) for different numbers of fragments (x-axis) using the flap endonuclease-mediated nucleic acid assembly, comparative 1, and comparative 2 methods.

FIGS. 20A-20B are assembly error plots for flap endonuclease-mediated nucleic acid assembly, comparative 1 assembly, and comparative 2 assembly.

Figure 20C is a graph of percentage of total counts (y-axis) relative to assemblies comparing endonuclease-mediated nucleic acid assembly, comparative 1 assembly, and comparative 2 assembly (x-axis).

FIG. 21A is a graph of assembled constructs before and after PCR amplification for various numbers of inserts.

Figure 21B is a graph of the percentage of total counts (y-axis) for different numbers of fragments (x-axis) relative to assemblies using the flap endonuclease-mediated nucleic acid assembly, comparative 1 and comparative 2 methods.

Figure 21C is a graph of nucleic acid assembly, population CFU percentage, and NGS results using flap endonuclease mediation.

FIG. 21D is a graph of the percentage of distribution of correctly assembled constructs and incorrectly assembled or incorrect constructs.

FIG. 22A is a plot of the observed frequency (y-axis) per 5,400 compared to various GC classes (x-axis) for the amplified oligonucleotide population prior to assembly.

Figure 22B is a graph of the observed frequency per 5,400 (y-axis) compared to various GC classes (x-axis) for a population of oligonucleotides assembled by flap endonuclease-mediated nucleic acid assembly.

FIG. 23 is a graph of gene level results from multiple gene assembly reactions.

Detailed Description

Definition of

Throughout this disclosure, various embodiments are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of any embodiment. Thus, unless the context clearly dictates otherwise, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range which are to the nearest tenth of the unit of the lower limit. For example, description of a range such as from 1 to 6 should be considered to have explicitly disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual values within that range, e.g., 1.1, 2, 2.3, 5, and 5.9. This applies regardless of the breadth of the range. The upper and lower limits of these intermediate ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Unless the context clearly dictates otherwise, when a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of any embodiment. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and/or "comprising," when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

Unless specified otherwise, or apparent from context, the term "nucleic acid" as used herein encompasses double-or triple-stranded nucleic acids as well as single-stranded molecules. In double-stranded or triple-stranded nucleic acids, the nucleic acid strands need not be coextensive (i.e., the double-stranded nucleic acid need not be double-stranded along the entire length of both strands). When provided, nucleic acid sequences are listed in a 5 'to 3' orientation unless otherwise indicated. The methods described herein provide for the generation of isolated nucleic acids. The methods described herein additionally provide for the generation of isolated and purified nucleic acids. Reference herein to a "nucleic acid" can include at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000 or more bases in length. Also, provided herein are methods of synthesizing any number of nucleotide sequences encoding polypeptide segments, including sequences encoding non-ribosomal peptides (NRPs), sequences encoding: non-ribonucleopeptide synthetase (NRPS) modules and synthetic variants, polypeptide segments of other modular proteins such as antibodies, polypeptide segments from other protein families, including non-coding DNA or RNA, such as regulatory sequences, e.g., promoters, transcription factors, enhancers, siRNA, shRNA, RNAi, miRNA, nucleolar small RNA derived from microrna, or any functional or structural DNA or RNA unit of interest. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, intergenic DNA, locus(s) defined by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (sirna), short hairpin RNA (shrna), micro-RNA (mirna), nucleolar small RNA, ribozymes, complementary DNA (cdna), which is a DNA-rendered form of mRNA, typically obtained by reverse transcription of messenger RNA (mRNA) or by amplification; DNA molecules, genomic DNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers, produced synthetically or by amplification. The cDNA encoding the genes or gene fragments referred to herein may comprise at least one region encoding an exon sequence without intervening intron sequences in the genomic equivalent sequence.

As used herein, the term "about" with respect to a number or range of numbers should be understood to mean the number and +/-10% of the number or value for the range, or 10% below the lower limit to 10% above the upper limit, as specified, unless otherwise indicated or apparent from the context.

Primers referred to as "universal primers" in the exemplary workflows referred to herein are short polynucleotides that recognize a primer binding site common to multiple DNA fragments. However, these workflows are not limited to the use of only universal primers, and additionally or alternatively fragment-specific primers can be incorporated. In addition, although the exemplary workflows described herein refer to the assembly of gene fragments, they are not so limited and are generally applicable to the assembly of longer nucleic acids.

Seamless nucleic acid assembly

Provided herein are methods of nucleic acid assembly with improved efficiency and accuracy. Further provided herein are methods of assembling nucleic acids into long genes. Polynucleotides described herein are assembled into longer nucleic acids by an assembly method comprising an endonuclease or exonuclease, optionally in combination with additional enzymes.

An exemplary method of assembling nucleic acids using flap endonucleases is depicted in fig. 1A. Flap endonuclease-mediated nucleic acid assembly was performed using first gene segment 127 and second gene segment 131. The bottom strand of first gene segment 127 is designed to comprise, from 5 'to 3', a first universal primer binding sequence 107a, a homologous sequence 103, an insertion sequence 108, and a second universal primer binding sequence 107 b. The top strand of the second gene segment 131 is designed to comprise, from 5 'to 3', a first universal primer binding sequence 107c, a homologous sequence 105, an insertion sequence 110, and a second universal primer binding sequence 107 d. The first gene fragment 127 and the second gene fragment 131 are contacted with a reaction mixture comprising an exonuclease, a flap endonuclease, a polymerase, and a ligase. Exonuclease digests 1093' end to expose the homologous site, generating fragment 133. In some cases, the exonuclease is exonuclease III. Flap endonuclease cleaves 1115' the flap, generating fragment 135. In some cases, the flap endonuclease is flap endonuclease 1 (FEN-1). The polymerase fills in the gap 113 and leaves a nick, thereby generating fragment 137. The ligase then seals 115 the nick, thereby creating fragment 139.

An exemplary method of assembling nucleic acids using flap endonucleases and a bridge assembly method is depicted in fig. 1B. Flap endonuclease-mediated nucleic acid assembly is performed using double-stranded nucleic acid bridge 151, first gene segment 155, and second gene segment 157. Double-stranded nucleic acid bridge 151 comprises a first universal primer binding sequence 153a, a first homologous sequence 155a that is homologous to first gene segment 155, a second homologous sequence 157a that is homologous to second gene segment 157, and a second universal primer binding sequence 153 b. Double-stranded nucleic acid bridge 151, first gene segment 155, and second gene segment 157 are contacted with a reaction mixture comprising an exonuclease, a flap endonuclease, a polymerase, and a ligase. Exonuclease digests 1593' end to expose the homology site, resulting in fragment 169. In some cases, the exonuclease is exonuclease III. The polymerase fills in gap 161 and leaves a cut, thereby creating fragment 171. The flap endonuclease cut 1655' flap, generating fragment 173. In some cases, the flap endonuclease is flap endonuclease 1 (FEN-1). The ligase then seals 167 the nick, thereby generating fragment 175. In some cases, the ligase is ampligase.

Provided herein are enzymatically mediated nucleic acid assembly methods. In some cases, the enzymatically mediated assembly of nucleic acids comprises the addition of homologous sequences to a gene fragment. In some cases, de novo synthesized gene fragments already contain homologous sequences. In some cases, the enzymatically mediated assembly of nucleic acids comprises the use of a mixture of enzymes. In some cases, the enzyme mixture comprises an endonuclease. In some cases, the enzyme mixture optionally comprises an exonuclease, a polymerase, or a ligase. In some cases, the enzyme mixture comprises an exonuclease, an endonuclease, a polymerase, and a ligase. In some cases, the enzyme mixture comprises an endonuclease, a polymerase, and a ligase. In some cases, the endonuclease is a flap endonuclease. In some cases, enzymatically mediated nucleic acid assembly results in increased efficiency. In some cases, the enzyme mixture comprises an enzyme that is not a restriction enzyme. In some cases, the enzyme mixture comprises an enzyme that is a structure-specific enzyme. In some cases, the enzyme mixture comprises enzymes that are structure-specific enzymes rather than sequence-specific enzymes.

Provided herein are methods in which a site-specific base excision reagent comprising one or more enzymes is used as a cleavage agent that cleaves only a single strand of double-stranded DNA at the cleavage site. Many repair enzymes are suitable for creating such incisions, either alone or in combination with other agents. An exemplary list of repair enzymes is provided in table 1. According to various embodiments, homologues or non-natural variants of repair enzymes are also used, including those in table 1. Any repair enzyme used in accordance with the methods and compositions described herein can be naturally occurring, recombinant, or synthetic. In some cases, the DNA repair enzyme is a naturally or in vitro produced chimeric protein having one or more activities. In various embodiments, the cleavage agent has enzymatic activity, including an enzyme mixture comprising one or more of a nicking endonuclease, an AP endonuclease, a glycosylase, and a lyase, involved in base excision repair.

Repair enzymes are found in prokaryotic and eukaryotic cells. Some enzymes having applicability herein have glycosylase and AP endonuclease activity in one molecule. AP endonucleases are classified according to their cleavage site. Class I and class II AP endonucleases cleave DNA at phosphate groups on the 3' and 5' side of the abasic site, leaving 3' -OH and 5-phosphate ends. Class III and IV AP endonucleases also cleave DNA at phosphate groups on the 3 'and 5' side of the abasic site, but they produce 3 '-phosphate and 5' -OH. Examples of polynucleotide cleaving enzymes used include DNA repair enzymes, which are listed in table 1.

TABLE 1 DNA repair enzymes

Provided herein are enzymatically-mediated nucleic acid assembly methods, wherein a gene fragment or gene for assembly comprises a homologous sequence. In some cases, the homologous sequence comprises at least or about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more than 100 base pairs. In some cases, the number of base pairs is 40 base pairs. In some cases, the number of base pairs has a range of about 5 to 100, 10 to 90, 20 to 80, 30 to 70, or 40 to 60 base pairs.

Provided herein are enzymatically-mediated nucleic acid assembly methods, wherein the gene segments or genes used for assembly do not comprise homologous sequences. In some cases, for de novo synthesized gene fragments without homologous sequences, the enzymatically-mediated nucleic acid assembly method involves assembly using nucleic acid bridges. In some cases, the nucleic acid bridge comprises DNA or RNA. In some cases, the nucleic acid bridge comprises DNA. In some cases, the nucleic acid bridge is double-stranded. In some cases, the nucleic acid bridge is single stranded.

Provided herein are enzymatically mediated nucleic acid assembly methods using a nucleic acid bridge, wherein the nucleic acid bridge comprises one or more universal primer binding sequences. In some cases, the nucleic acid bridge comprises at least or about 1,2, 3, 4,5, 6, 7, 8, or more than 8 universal primer binding sequences. In some cases, the nucleic acid bridge further comprises a homologous sequence. In some cases, the homologous sequence is homologous to a de novo synthesized gene fragment. In some cases, the nucleic acid bridge further comprises one or more homologous sequences. For example, the nucleic acid bridge comprises one or more homologous sequences that are homologous to different de novo synthesized gene fragments. In some cases, the nucleic acid bridge comprises 1,2, 3, 4,5, 6, 7, 8, 9, 10, or more than 10 homologous sequences. In some cases, the homologous sequence comprises at least or about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more than 100 base pairs. In some cases, the number of base pairs is 40 base pairs. In some cases, the number of base pairs is 50 base pairs. In some cases, the number of base pairs has a range of about 5 to 100, 10 to 90, 20 to 80, 30 to 70, or 40 to 60 base pairs.

Provided herein are enzymatically mediated nucleic acid assembly methods in which double-stranded nucleic acids are contacted with an enzyme having exonuclease activity. In some cases, the exonuclease has 3' exonuclease activity. Exemplary exonucleases having 3' exonuclease activity include, but are not limited to, exonuclease I, exonuclease III, exonuclease V, exonuclease VII, and exonuclease T. In some cases, the exonuclease has 5' exonuclease activity. Exemplary exonucleases having 5' exonuclease activity include, but are not limited to, exonuclease II, exonuclease IV, exonuclease V, exonuclease VI, exonuclease VII, exonuclease VIII, T5 exonuclease and T7 exonuclease. In some cases, the exonuclease is exonuclease iii (exoiii). Exonucleases include wild-type exonucleases and derivatives, chimeras and/or mutants thereof. Mutant exonucleases include enzymes that comprise one or more mutations, insertions, deletions or any combination thereof within the amino acid or nucleic acid sequence of an exonuclease.

In some cases, the exonuclease is used at a temperature optimal for enzyme activity, e.g., at a temperature in the range of about 25-80 ℃, 25-70 ℃, 25-60 ℃, 25-50 ℃, or 25-40 ℃. In some cases, the temperature is about 37 ℃. In some cases, the temperature is about 50 ℃. In some cases, the temperature is about 55 ℃. In some cases, the temperature is about 65 ℃. In some cases, the temperature is at least or about 15 ℃,20 ℃, 25 ℃,30 ℃, 35 ℃,40 ℃, 45 ℃,50 ℃, 55 ℃,60 ℃, 65 ℃,70 ℃, 75 ℃,80 ℃ or greater than 80 ℃.

In some cases, the enzymatically-mediated nucleic acid assembly method does not include the use of an exonuclease. In some cases, the enzymatically mediated nucleic acid assembly method comprises the use of an exonuclease. In some cases, one or more exonucleases are used. For example, at least or about 1,2, 3, 4,5, 6, or more than 6 exonucleases are used. In some cases, the exonuclease has 5 'to 3' exonuclease activity. In some cases, the exonuclease has 3 'to 5' exonuclease activity. In some cases, the method comprises contacting the double-stranded DNA with an endonuclease. In some cases, the endonuclease is a flap endonuclease. In some cases, the method comprises contacting the double-stranded DNA with a flap endonuclease, a ligase, or a polymerase. In some cases, the flap endonuclease is flap endonuclease 1.

Provided herein are methods in which double-stranded nucleic acids are treated with an enzyme having endonuclease activity. In some cases, the endonuclease has 5' nuclease activity. In some cases, the endonuclease has 3' nuclease activity. In some cases, the endonuclease is a flap endonuclease. In some cases, the flap endonuclease has 5' nuclease activity. In some cases, the flap endonuclease is a member of the 5' -nuclease family of enzymes. Exemplary 5' -nucleases include, but are not limited to, flap endonuclease 1, exonuclease 1, xeroderma pigmentosum complementation group g (xpg), Dna2, and nick endonuclease 1(GEN 1). In some cases, the flap endonuclease is flap endonuclease 1. In some cases, the flap endonuclease has 3' nuclease activity. Exemplary flap endonucleases having 3' nuclease activity include, but are not limited to, RAG1, RAG2, and MUS 81. In some cases, the flap endonuclease is an archaea, bacterial, yeast, plant, or mammalian flap endonuclease. Exemplary 5 'nucleases and 3' nucleases can be found in table 2.

TABLE 2 exemplary nucleases

In some cases, the endonuclease is used at a temperature optimal for enzyme activity, e.g., at a temperature of 25-80 ℃, 25-70 ℃, 25-60 ℃, 25-50 ℃, or 25-40 ℃. In some cases, the temperature is about 50 ℃. In some cases, the temperature is about 55 ℃. In some cases, the temperature is about 65 ℃. In some cases, the temperature is at least or about 15 ℃,20 ℃, 25 ℃,30 ℃, 35 ℃,40 ℃, 45 ℃,50 ℃, 55 ℃,60 ℃, 65 ℃,70 ℃, 75 ℃,80 ℃ or greater than 80 ℃. In some cases, the endonuclease is a thermostable endonuclease. Thermostable endonucleases can include endonucleases that are functional at temperatures of at least or about 60 ℃, 65 ℃,70 ℃, 75 ℃,80 ℃ or above 80 ℃. In some cases, the endonuclease is a flap endonuclease. In some cases, the flap endonuclease is a thermostable flap endonuclease.

Provided herein are nucleic acid assembly methods, wherein the ratio of the endonuclease to the exonuclease is from about 0.1:1 to about 1: 5. In some cases, the endonuclease is a flap endonuclease. In some cases, the ratio of endonuclease to exonuclease is at least or about 0.2:1, 0.25:1, 0.5:1, 0.75:1, 1:1.5, 1:2, 1:3, 1:4, 1:5, or more than 1: 5. In some cases, the ratio of endonuclease to exonuclease is at least or about 1:1, 1:0.9, 1:0.85, 1:0.8, 1:0.75, 1:0.7, 1:0.65, 1:0.6, 1:0.55, 1:0.5, 1:0.45, 1:0.4, 1:0.35, 1:0.3, 1:0.25, 1:0.2, 1:0.15, 1:0.1, or less than 1: 0.1.

Provided herein are nucleic acid assembly methods comprising an exonuclease, wherein the concentration of the exonuclease is from about 0.1U to about 20U or higher. For example, the concentration of the exonuclease is at least or about 0.1U, 0.25U, 0.5U, 0.75U, 1U, 1.6U, 2U, 3U, 4U, 5U, 6U, 7U, 8U, 9U, 10U, 12U, 14U, 16U, 18U, 20U, or more than 20U. In some cases, the concentration of the exonuclease is in the range of about 0.5U to about 1.0U. In some cases, the concentration of the exonuclease is from about 1.0U to about 2.0U. In some cases, the concentration of the exonuclease is about 1.6U. In some cases, the concentration of the exonuclease is about 5.0U. In some cases, the concentration of the exonuclease ranges from about 0.1U to 20U, 0.25U to 18U, 0.5U to 16U, 0.75U to 14U, 1U to 12U, 2U to 10U, 3U to 9U, or 4U to 8U.

The enzymatically-mediated nucleic acid assembly methods described herein can include an endonuclease, wherein the concentration of the endonuclease is from about 0.25U to about 12U or higher. In some cases, the endonuclease is a flap endonuclease. Exemplary concentrations of the endonuclease include, but are not limited to, at least or about 0.25U, 0.5U, 0.75U, 1U, 2U, 3U, 4U, 5U, 6U, 7U, 8U, 9U, 10U, 11U, 12U, or more than 12U. In some cases, the concentration of the endonuclease is about 0.32U. In some cases, the concentration of the endonuclease is about 1.6U. In some cases, the concentration of the endonuclease is in the range of about 0.32U to about 4.8U. In some cases, the concentration of the endonuclease is in a range of about 0.25U to 12U, 0.5U to 11U, 0.75U to 10U, 1U to 9U, 2U to 8U, 3U to 7U, or 4U to 6U.

Provided herein are enzymatically mediated nucleic acid assembly methods in which double-stranded nucleic acids are mixed with a polymerase. In some cases, the polymerase is a DNA polymerase. In some cases, the polymerase is a high fidelity polymerase. High fidelity polymerases may include polymerases that result in the accurate replication or amplification of a template nucleic acid. In some cases, the DNA polymerase is a thermostable DNA polymerase. The DNA polymerase may be from any DNA polymerase family including, but not limited to, group a polymerases, group B polymerases, group C polymerases, group D polymerases, group X polymerases, and group Y polymerases. In some cases, the DNA polymerase is from a genus including, but not limited to, Thermus (Thermus), Bacillus (Bacillus), Pyrococcus (Thermococcus), Pyrococcus (Pyrococcus), Aeropyrum (Aeropyrum), liquidproducing bacteria (Aquifex), Sulfolobus (Sulfolobus), pyretobacter (Pyrolobus), or Methanopyrus (Methanopyrus).

The polymerases used in amplification reactions described herein can have a variety of enzymatic activities. Polymerase in the textThe methods of the invention are used, for example, to extend a primer to produce an extension product. In some cases, the DNA polymerase has 5 'to 3' polymerase activity. In some cases, the DNA polymerase has 3 'to 5' exonuclease activity. In some cases, the DNA polymerase has proofreading activity. Exemplary polymerases include, but are not limited to, DNA polymerase (I, II or III), T4 DNA polymerase, T7 DNA polymerase, Bst DNA polymerase, Bca polymerase, Vent DNA polymerase, Pfu DNA polymerase, and Taq DNA polymerase. Non-limiting examples of thermostable DNA polymerases include, but are not limited to, Taq,DNA polymerase,High Fidelity DNA polymerase,DNA polymerase, expanded High Fidelity polymerase, HotTub polymerase, Pwo polymerase, Tfl polymerase, Tli polymerase, UlTma polymerase, Pfu polymerase, KOD DNA polymerase, JDF-3DNA polymerase, PGB-D DNA polymerase, Tgo DNA polymerase, Pyrolobus furaria DNA polymerase, Vent polymerase and Deep Vent polymerase.

Described herein are methods comprising a DNA polymerase, wherein the concentration of the DNA polymerase is from about 0.1U to about 2U, or greater than 2U. In some cases, the concentration of the DNA polymerase is about 0.1U. In some cases, the concentration of the DNA polymerase is about 0.2U. In some cases, the concentration of the DNA polymerase is about 0.01U. In some cases, the concentration of the DNA polymerase is in a range of at least or about 0.005U to 2U, 0.005U to 1U, 0.005U to.5U, 0.01U to 1U, 0.1U to 0.5U, 0.1U to 1U, 0.1U to 1.5U, 0.1U to 2U, 0.5U to 1.0U, 0.5U to 1.5U, 0.5U to 2U, 1U to 1.5U, 1.0U to 2.0U, or 1.5U to 2U.

The DNA polymerase for use in the methods described herein is used at a temperature optimal for enzyme activity, e.g., at a temperature of 25-80 ℃, 25-70 ℃, 25-60 ℃, 25-50 ℃ or 25-40 ℃. In some cases, the temperature is about 50 ℃. In some cases, the temperature is about 55 ℃. In some cases, the temperature is about 65 ℃. In some cases, the temperature is at least or about 15 ℃,20 ℃, 25 ℃,30 ℃, 35 ℃,40 ℃, 45 ℃,50 ℃, 55 ℃,60 ℃, 65 ℃,70 ℃, 75 ℃,80 ℃ or greater than 80 ℃.

The methods described herein for enzymatically mediated nucleic acid assembly can include an amplification reaction, wherein the amplification reaction comprises a universal primer binding sequence. In some cases, the universal primer binding sequence is capable of binding to the same 5 'or 3' primer. In some cases, the universal primer binding sequence is shared between multiple target nucleic acids in the amplification reaction.

Provided herein are enzymatically mediated nucleic acid assembly methods in which double-stranded nucleic acids are treated with a ligase. A ligase as described herein may serve to ligate nucleic acid fragments. For example, the ligase is used to join adjacent 3 '-hydroxylated and 5' -phosphorylated ends of DNA. Ligases include, but are not limited to, E.coli ligase, T4 ligase, mammalian ligases (e.g., DNA ligase I, DNA ligase II, DNA ligase III, DNA ligase IV), thermostable ligases, and fast ligases. In some cases, the ligase is a thermostable ligase. In some cases, the ligase is Ampligase.

The concentration of ligase may vary. In some cases, the concentration of ligase is in the range of about 0U to about 2U. An exemplary concentration of ligase is approximately 0.5U. In some cases, the concentration of ligase is about 1.0U. In some cases, the concentration of ligase is about 5.0U. In some cases, the concentration of ligase is in a range of at least or about 0U to 0.25U, 0U to 0.5U, 0U to 1U, 0U to 1.5U, 0U to 2U, 0.25U to 0.5U, 0.25U to 1.0U, 0.25U to 1.5U, 0.25U to 2.0U, 0.5U to 1.0U, 0.5U to 1.5U, 0.5U to 2.0U, 1.0U to 1.5U, 1.0U to 2.0U, 1.5U to 2.0U, 2.0U to 4.0U, 4.0U to 6.0U, 4.0U to 8.0U, 6.0U to 10.0U.

In some cases, the ligase is used at a temperature optimal for enzyme activity, e.g., at a temperature of 25-80 ℃, 25-70 ℃, 25-60 ℃, 25-50 ℃, or 25-40 ℃. In some cases, the temperature is about 50 ℃. In some cases, the temperature is about 55 ℃. In some cases, the temperature is about 65 ℃. In some cases, the temperature is at least or about 15 ℃,20 ℃, 25 ℃,30 ℃, 35 ℃,40 ℃, 45 ℃,50 ℃, 55 ℃,60 ℃, 65 ℃,70 ℃, 75 ℃,80 ℃ or greater than 80 ℃.

Provided herein are methods for enzymatically mediated nucleic acid assembly, wherein a plurality of gene fragments are assembled. In some cases, the gene segments are assembled continuously or sequentially. In some cases, the gene fragments are assembled into vectors. In some cases, gene fragment assembly is used for long linear gene assembly. In some cases, the number of gene segments is at least or about 2, 3, 4,5, 6, 7, 8, 9, 10, or more than 10 gene segments. In some cases, the number of gene segments is at least or about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 gene segments. In some cases, the number of gene segments is in a range of about 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 2 to 3, 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 3 to 4, 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, 3 to 10, 4 to 5,4 to 6, 4 to 7, 4 to 8,4 to 9, 4 to 10, 5 to 6, 5 to 7, 5 to 8, 5 to 9, 5 to 10, 6 to 7, 6 to 8, 6 to 9, 6 to 10, 7 to 8, 7 to 9, 7 to 10, 8 to 9, 8 to 10, or 9 to 10. In some cases, the number of gene segments is about 1 to about 20, about 2 to about 18, about 3 to about 17, about 4 to about 16, about 6 to about 14, or about 8 to about 12.

Provided herein are methods for enzymatically mediated nucleic acid assembly, wherein the ratio of assembled gene fragments is about 0.2:1, 0.25:1, 0.5:1, 0.75:1, 1:1.5, 1:2, 1:3, 1:4, 1:5, or greater than 1: 5. For example, if two gene segments are assembled, the ratio of the first gene segment to the second gene segment is 1:1. In some cases, the ratio of the first gene segment to the second gene segment is at least or about 1:1, 1:0.9, 1:0.85, 1:0.8, 1:0.75, 1:0.7, 1:0.65, 1:0.6, 1:0.55, 1:0.5, 1:0.45, 1:0.4, 1:0.35, 1:0.3, 1:0.25, 1:0.2, 1:0.15, 1:0.1, or less than 1: 0.1.

The methods described herein for enzymatically mediated nucleic acid assembly can comprise assembling one or more gene segments into a vector, wherein the ratio of the one or more gene segments to the vector is varied. In some cases, the ratio of the one or more gene segments to the vector is at least or about 0.2:1, 0.25:1, 0.5:1, 0.75:1, 1:1.5, 1:2, 1:3, 1:4, 1:5, or more than 1: 5. In some cases, the ratio of the one or more gene fragments to the vector is at least or about 1:1, 1:0.9, 1:0.85, 1:0.8, 1:0.75, 1:0.7, 1:0.65, 1:0.6, 1:0.55, 1:0.5, 1:0.45, 1:0.4, 1:0.35, 1:0.3, 1:0.25, 1:0.2, 1:0.15, 1:0.1, or less than 1: 0.1.

A method for enzymatically mediated nucleic acid assembly as described herein can comprise assembling a population of oligonucleotides for assembly into a vector. In some cases, overlap extension PCR is performed to assemble a population of oligonucleotides. In some cases, the population of oligonucleotides comprises at least or about 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, or more than 200 oligonucleotides. In some cases, a population of oligonucleotides is assembled to generate a long nucleic acid comprising at least or about 50, 100, 200, 250300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1300, 1400, 1500, 1600, 1700, 1800, 2000, 2400, 2600, 2800, 3000, 3200, 3400, 3600, 3800, 4000, 4200,4400, 4600, 4800, 5000, 6000, 7000, 8000, 9000, 10000, or more than 10000 bases.

Methods for enzymatically mediated nucleic acid assembly as described herein can include multiplex gene assembly. In some cases, multiple sequences are assembled in a single reaction. In some cases, at least or about 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, or more than 200 sequences are assembled in a single reaction. In some cases, sequences assembled by multiplex gene assembly are inserted into a vector.

Methods for enzymatically mediated nucleic acid assembly can include assembling one or more gene fragments using a nucleic acid bridge, wherein the ratio of the one or more gene fragments to the nucleic acid bridge is varied. In some cases, the ratio of the one or more gene fragments to the nucleic acid bridge is at least or about 0.2:1, 0.25:1, 0.5:1, 0.75:1, 1:1.5, 1:2, 1:3, 1:4, 1:5, or more than 1: 5. In some cases, the ratio of the one or more gene fragments to the nucleic acid bridge is at least or about 1:1, 1:0.9, 1:0.85, 1:0.8, 1:0.75, 1:0.7, 1:0.65, 1:0.6, 1:0.55, 1:0.5, 1:0.45, 1:0.4, 1:0.35, 1:0.3, 1:0.25, 1:0.2, 1:0.15, 1:0.1, or less than 1: 0.1.

Provided herein are methods for enzymatically mediated nucleic acid assembly of gene fragments, wherein the total size of the number of assembled gene fragments is at least or about 50, 100, 200, 250300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1300, 1400, 1500, 1600, 1700, 1800, 2000, 2400, 2600, 2800, 3000, 3200, 3400, 3600, 3800, 4000, 4200,4400, 4600, 4800, 5000, 6000, 7000, 8000, 9000, 10000, or more than 10000 bases. In some cases, the total size of the number of assembled gene fragments ranges from about 300 to 1,000, 300 to 2,000, 300 to 3,000, 300 to 4,000, 300 to 5,000, 300 to 6,000, 300 to 7,000, 300 to 8,000, 300 to 9,000, 300 to 10,000, 1,000 to 2,000, 1,000 to 3,000, 1,000 to 4,000, 1,000 to 5,000, 1,000 to 6,000, 1,000 to 7,000, 1,000 to 8,000, 1,000 to 9,000, 1,000 to 10,000, 2,000 to 3,000, 2,000 to 4,000, 2,000 to 5,000, 2,000 to 6,000, 2,000 to 7,000, 2,000 to 8,000, 2,000 to 9,000, 2,000 to 10,000, 3,000 to 4,000, 3,000 to 5,000, 6,000, 2,000 to 7,000, 2,000 to 8,000, 2,000 to 9,000, 6,000 to 7,000, 6,000 to 8,000, 6,000 to 8,000, 6,000, or 8,000 to 8,000.

The methods described herein that involve enzymatically mediated nucleic acid assembly result in a high percentage of correct assembly. In some cases, the percentage of correct assembly is at least or about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or more than 99%. In some cases, the percentage of average correct assembly is at least or about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or more than 99%. In some cases, the percentage of correct assembly is 100%.

The methods as described herein that include enzymatically mediated nucleic acid assembly result in a low percentage of misassemblies. In some cases, the percent mis-assembly rate is at most 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60%. In some cases, the percent mis-assembly rate is from about 1% to about 25%, from about 5% to about 20%, or from about 10% to about 15%. In some cases, the average mis-assembly rate is at most 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60%. In some cases, the average mis-assembly rate is from about 1% to about 25%, from about 5% to about 20%, or from about 10% to about 15%.

The methods described herein that include enzymatically mediated nucleic acid assembly result in increased efficiency. In some cases, efficiency is measured by the number of colony forming units. In some cases, the methods described herein result in at least or about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 12000, 14000, 16000, 18000, 20000, 25000, 30000, 35000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, or more than 100000 colony forming units.

System for nucleic acid synthesis and seamless assembly

Polynucleotide synthesis

Provided herein are methods for seamless assembly of nucleic acids after production of polynucleotides by de novo synthesis by the methods described herein. An exemplary workflow can be seen in fig. 2. A computer-readable input file comprising a nucleic acid sequence is received. Instructions for processing a nucleic acid sequence by a computer to generate a synthetic polynucleotide sequence or a plurality of polynucleotide sequences that collectively encode the nucleic acid sequence. Instructions are transmitted to the material deposition device 203 for synthesizing the plurality of polynucleotides based on the plurality of nucleic acid sequences. The material deposition device 203, such as a polynucleotide synthesizer, is designed to release reagents in a stepwise manner such that multiple polynucleotides are extended in parallel one residue at a time to generate an oligomer having a predetermined nucleic acid sequence. The material deposition apparatus 203 generates oligomers on an array 205 comprising a plurality of clusters 207 of loci for polynucleotide synthesis and extension. However, the array need not have the loci organized in clusters. For example, the seats may be evenly distributed throughout the array. The slave polynucleotide is synthesized and removed from the plate and the assembly reaction is started in the collection chamber 209, then a population 211 of longer polynucleotides is formed. The collection chamber may comprise multiple surfaces (e.g., top and bottom surfaces) or a sandwich of wells or channels that accommodate the transferred material from the synthetic surface. The de novo polynucleotide may also be synthesized and removed from the plate to form a population 211 of longer polynucleotides. The population 211 of longer polynucleotides may then be divided into droplets or subjected to PCR. The population 211 of longer polynucleotides is then subjected to nucleic acid assembly by flap endonuclease-mediated nucleic acid assembly 213.

Provided herein are systems for seamless assembly of nucleic acids following production of polynucleotides by de novo synthesis by the methods described herein. In some cases, the system includes a computer, a material deposition device, a surface, and a nucleic acid assembly surface. In some cases, the computer includes a readable input file having a nucleic acid sequence. In some cases, the computer processes the nucleic acid sequence to generate instructions for synthesizing a polynucleotide sequence or a plurality of polynucleotide sequences that collectively encode the nucleic acid sequence. In some cases, the computer provides instructions to the material deposition apparatus regarding synthesizing a plurality of polynucleotide sequences. In some cases, the material deposition device deposits nucleosides on a surface for an extension reaction. In some cases, the surface includes a seat for an extension reaction. In some cases, the locus is a spot, hole, micropore, channel or post (post). In some cases, the plurality of polynucleotide sequences is synthesized after the extension reaction. In some cases, the plurality of polynucleotide sequences are removed from the surface and prepared for nucleic acid assembly. In some cases, the nucleic acid assembly comprises a flap endonuclease-mediated nucleic acid assembly.

Provided herein are methods for polynucleotide synthesis involving phosphoramidite chemistry. In some cases, polynucleotide synthesis includes coupling bases with phosphoramidites. In some cases, polynucleotide synthesis includes coupling bases by depositing phosphoramidite under coupling conditions, wherein the same base is optionally deposited more than once with the phosphoramidite, i.e., double coupling. In some cases, polynucleotide synthesis includes capping of unreacted sites. In some cases, capping is optional. In some cases, polynucleotide synthesis comprises oxidation. In some cases, polynucleotide synthesis includes deblocking or detritylation. In some cases, polynucleotide synthesis comprises sulfurization. In some cases, polynucleotide synthesis comprises oxidation or sulfurization. In some cases, the substrate is washed, for example with tetrazole or acetonitrile, between one or each step during the polynucleotide synthesis reaction. The time range for any step in the phosphoramidite synthesis process includes less than about 2min, 1min, 50sec, 40sec, 30sec, 20sec, or 10 sec.

Polynucleotide synthesis using the phosphoramidite approach involves the subsequent addition of a phosphoramidite building block (e.g., a nucleoside phosphoramidite) to a growing polynucleotide chain to form a phosphite triester linkage. Phosphoramidite polynucleotide synthesis proceeds in the 3 'to 5' direction. Phosphoramidite polynucleotide synthesis allows for the controlled addition of one nucleotide to a growing nucleic acid strand in each synthesis cycle. In some cases, each synthesis cycle includes a coupling step. Phosphoramidite coupling involves the formation of a phosphite triester bond between an activated nucleoside phosphoramidite and a nucleoside bound to a substrate (e.g., via a linker). In some cases, the nucleoside phosphoramidite is provided to an activated substrate. In some cases, the nucleoside phosphoramidite is provided to a substrate with an activator. In some cases, the nucleoside phosphoramidite is provided to the substrate in an excess of 1.5, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100-fold or more relative to the nucleoside bound to the substrate. In some cases, the addition of the nucleoside phosphoramidite is performed in an anhydrous environment (e.g., in anhydrous acetonitrile). After addition of the nucleoside phosphoramidite, the substrate is optionally washed. In some cases, the coupling step is repeated one or additional times, optionally with a washing step between the addition of nucleoside phosphoramidite to the substrate. In some cases, a polynucleotide synthesis method as used herein comprises 1,2, 3, or more sequential coupling steps. In many cases, prior to coupling, the substrate-bound nucleoside is deprotected by removal of a protecting group, wherein the protecting group acts to prevent polymerization. A common protecting group is 4, 4' -Dimethoxytrityl (DMT).

Following coupling, the phosphoramidite polynucleotide synthesis method optionally includes a capping step. In the capping step, the growing polynucleotide is treated with a capping agent. The capping step can be used to block unreacted substrate-bound 5' -OH groups after coupling to prevent further chain extension, thereby preventing formation of polynucleotides with internal base deletions. Furthermore, phosphoramidites activated with 1H-tetrazole react to a very small extent with the O6 position of guanosine. Without being bound by theory, in the use of I₂After water oxidation, this by-product (possibly migrating via O6-N7) can undergo depurination. The apurinic site may end up being cleaved during the final deprotection of the polynucleotide, thereby reducing the yield of the full-length product. The O6 modification can be carried out by using I₂The water is removed by treatment with a capping reagent prior to oxidation. In some cases, including a capping step during polynucleotide synthesis reduces the error rate compared to synthesis without capping. As an example, the capping step comprises treating the polynucleotide bound to the substrate with a mixture of acetic anhydride and 1-methylimidazole. After the capping step, the substrate is optionally washed.

In some cases, the growing nucleic acid bound to the substrate is oxidized after the addition of the nucleoside phosphoramidite, and optionally after capping and one or more washing steps. The oxidation step involves oxidation of the phosphite triester to a tetracoordinated phosphotriester, a protected precursor to the naturally occurring phosphodiester internucleoside linkage. In some cases, oxidation of the growing polynucleotide is achieved by treatment with iodine and water, optionally in the presence of a weak base (e.g., pyridine, lutidine, collidine). The oxidation can be carried out under anhydrous conditions using, for example, tert-butyl hydroperoxide or (1S) - (+) - (10-camphorsulfonyl) -oxaziridine (CSO). In some methods, a capping step is performed after the oxidizing. The second capping step allows the substrate to dry, since residual water from oxidation, which may persist, may inhibit subsequent coupling. After oxidation, the substrate and the growing polynucleotide are optionally washed. In some cases, the oxidation step is replaced with a sulfurization step to obtain a polynucleotide phosphorothioate, wherein any capping step may be performed after sulfurization. A number of reagents are capable of effective sulfur transfer, including but not limited to 3- (dimethylaminomethylene) amino) -3H-1,2, 4-dithiazole-3-thione, DDTT, 3H-1, 2-benzodithiolan-3-one 1, 1-dioxide (also known as Beaucage reagent), and N, N, N' -tetraethylthiuram disulfide (TETD).

To allow subsequent cycles of nucleoside incorporation to occur through coupling, the protected 5' end of the growing polynucleotide bound to the substrate is removed, allowing the primary hydroxyl group to be reactive with the next nucleoside phosphoramidite. In some cases, the protecting group is DMT, and deblocking is performed with trichloroacetic acid in dichloromethane. Performing detritylation for extended periods of time or detritylation using stronger acid solutions than the recommended acid solutions can result in increased depurination of the polynucleotide bound to the solid support, thus reducing the yield of the desired full length product. The methods and compositions of the invention described herein provide controlled deblocking conditions to limit undesirable depurination reactions. In some cases, the substrate-bound polynucleotide is washed after deblocking. In some cases, efficient washing after deblocking facilitates synthesis of polynucleotides with low error rates.

Polynucleotide synthesis methods generally comprise a series of iterative steps of: applying a protected monomer to an activated functionalized surface (e.g., a locus) to attach to an activated surface, a linker, or to a previously deprotected monomer; deprotecting the applied monomer to make it reactive with a subsequently applied protected monomer; and applying another protected monomer for attachment. One or more intermediate steps include oxidation or sulfidation. In some cases, one or more washing steps may precede or follow one or all of the steps.

Phosphoramidite-based polynucleotide synthesis methods involve a series of chemical steps. In some cases, one or more steps of a synthetic method involve reagent cycling, wherein one or more steps of the method include applying to the substrate a reagent useful for that step. For example, the reagents are cycled through a series of liquid phase deposition and vacuum drying steps. For substrates containing three-dimensional features such as wells, microwells, channels, etc., reagents optionally pass through one or more regions of the substrate via the wells and/or channels.

Polynucleotides synthesized using the methods and/or substrates described herein comprise at least about 20, 30, 40, 50, 60, 70, 75, 80, 90, 100, 120, 150, 200, 500, or more bases in length. In some cases, at least about 1pmol, 10pmol, 20pmol, 30pmol, 40pmol, 50pmol, 60pmol, 70pmol, 80pmol, 90pmol, 100pmol, 150pmol, 200pmol, 300pmol, 400pmol, 500pmol, 600pmol, 700pmol, 800pmol, 900pmol, 1nmol, 5nmol, 10nmol, 100nmol or more of the polynucleotide is synthesized in the locus. The methods of synthesizing polynucleotides on a surface provided herein allow for faster synthesis. As an example, at least 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 125, 150, 175, 200 or more nucleotides per hour are synthesized. Nucleotides include adenine, guanine, thymine, cytosine, uridine building blocks, or analogs/modified forms thereof. In some cases, the polynucleotide libraries are synthesized in parallel on a substrate. For example, a substrate comprising about or at least about 100, 1,000, 10,000, 100,000, 1,000,000, 2,000,000, 3,000,000, 4,000,000, or 5,000,000 resolved loci can support the synthesis of at least the same number of different polynucleotides, wherein polynucleotides encoding different sequences are synthesized at resolved loci.

Various suitable methods for generating high density polynucleotide arrays are known. In an exemplary workflow, a substrate surface layer is provided. In this example, the chemistry of the surface is altered to improve the polynucleotide synthesis process. The low surface energy regions are created to repel liquid while the high surface energy regions are created to attract liquid. The surface itself may be in the form of a planar surface or contain changes in shape, such as protrusions or pores that increase the surface area. In this workflow example, the selected high surface energy molecule serves the dual function of supporting DNA chemistry, as disclosed in international patent application publication WO/2015/021080, which is incorporated herein by reference in its entirety.

In situ preparation of polynucleotide arrays is performed on a solid support and multiple oligomers are extended in parallel using a single nucleotide extension process. The deposition device, such as a polynucleotide synthesizer, is designed to release reagents in a stepwise manner such that multiple polynucleotides are extended in parallel one residue at a time to generate an oligomer having a predetermined nucleic acid sequence. In some cases, the polynucleotide is cleaved from the surface at this stage. Cleavage includes, for example, gas cleavage with ammonia or methylamine.

Substrate

Devices used as polynucleotide synthesis surfaces may be in the form of substrates including, but not limited to, homogeneous array surfaces, patterned array surfaces, channels, beads, gels, and the like. Provided herein are substrates comprising a plurality of clusters, wherein each cluster comprises a plurality of loci that support polynucleotide attachment and synthesis. The term "locus" as used herein refers to a discrete region on a structure that provides support for extension of a polynucleotide encoding a single predetermined sequence from the surface. In some cases, the seat is on a two-dimensional surface (e.g., a substantially planar surface). In some cases, the seat is on a three-dimensional surface (e.g., a hole, a micro-hole, a channel, or a post). In some cases, the surface of the locus comprises a material that is activated and functionalized to attach at least one nucleotide for polynucleotide synthesis, or preferably, to attach a population of the same nucleotide for polynucleotide population synthesis. In some cases, a polynucleotide refers to a population of polynucleotides that encode the same nucleic acid sequence. In some cases, the surface of the substrate includes one or more surfaces of the substrate. The average error rate of polynucleotides synthesized within the libraries described herein using the provided systems and methods is typically less than 1/1000, less than about 1/2000, less than about 1/3000, or lower, typically without error correction.

Provided herein are surfaces that support the parallel synthesis of a plurality of polynucleotides having different predetermined sequences at addressable locations on a common support. In some cases, the substrate provides support for the synthesis of more than 50, 100, 200, 400, 600, 800, 1000, 1200, 1400, 1600, 1800, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1,000,000, 1,200,000, 1,400,000, 1,600,000, 1,800,000, 2,000,000, 2,500,000, 3,000,000, 3,500,000, 4,000,000, 4,500,000, 5,000,000, 10,000,000 or more different polynucleotides. In some cases, the surface provides support for synthesizing more than 50, 100, 200, 400, 600, 800, 1000, 1200, 1400, 1600, 1800, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1,000,000, 1,200,000, 1,400,000, 1,600,000, 1,800,000, 2,000,000, 2,500,000, 3,000,000, 3,500,000, 4,000,000, 4,500,000, 5,000,000, 10,000,000 or more polynucleotides encoding different sequences. In some cases, at least a portion of the polynucleotides have the same sequence or are configured to be synthesized with the same sequence. In some cases, the substrate provides a surface environment for growing polynucleotides having at least 80, 90, 100, 120, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more bases.

Provided herein are methods of synthesizing polynucleotides at different loci on a substrate, wherein each locus supports a synthetic polynucleotide population. In some cases, each locus supports the synthesis of a population of polynucleotides having a different sequence than the population of polynucleotides growing at another locus. In some cases, each polynucleotide sequence is synthesized with 1,2, 3, 4,5, 6, 7, 8, 9 or more redundancies at different loci within the same locus cluster on the surface used for polynucleotide synthesis. In some cases, the loci of the substrate are located within a plurality of clusters. In some cases, the substrate comprises at least 10, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 20000, 30000, 40000, 50000 or more clusters. In some cases, the substrate comprises more than 2,000, 5,000, 10,000, 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1,000,000, 1,100,000, 1,200,000, 1,300,000, 1,400,000, 1,500,000, 1,600,000, 1,700,000, 1,800,000, 1,900,000, 2,000,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1,000,000, 1,200,000, 1,400,000, 1,600,000, 1,800,000, 2,000,000, 2,500,000, 3,000,000, 3,500,000, 4,000, 4,500,000, 5,000, or 10,000 or more different seats. In some cases, the substrate comprises about 10,000 different seats. The amount of seats within a single cluster is different in different situations. In some cases, each cluster contains 1,2, 3, 4,5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 130, 150, 200, 300, 400, 500 or more seats. In some cases, each cluster contains about 50-500 loci. In some cases, each cluster contains about 100 and 200 loci. In some cases, each cluster contains approximately 100 and 150 loci. In some cases, each cluster contains about 109, 121, 130, or 137 loci. In some cases, each cluster contains about 19, 20, 61, 64, or more loci.

In some cases, the number of different polynucleotides synthesized on the substrate depends on the number of different loci available on the substrate. In some cases, the density of seats within a cluster of the substrateAt least or about 1,10, 25, 50, 65, 75, 100, 130, 150, 175, 200, 300, 400, 500, 1,000 or more seats/mm². In some cases, the substrate comprises 10-500, 25-400, 50-500, 100-500, 150-500, 10-250, 50-250, 10-200, or 50-200mm². In some cases, the distance between the centers of two adjacent seats within a cluster is about 10-500, about 10-200, or about 10-100 um. In some cases, the distance between the two centers of adjacent seats is greater than about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 um. In some cases, the distance between the centers of two adjacent seats is less than about 200, 150, 100, 80, 70, 60, 40, 30, 20, or 10 um. In some cases, each seat independently has a width of about 0.5, 1,2, 3, 4,5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 um. In some cases, each seat independently has a width of about 0.5-100, 0.5-50, 10-75, or 0.5-50 um.

In some cases, the density of clusters within the substrate is at least or about 1 cluster per 100mm²1 cluster/10 mm²1 cluster/5 mm²1 cluster/4 mm²1 cluster/3 mm²1 cluster/2 mm²1 cluster/1 mm²2 clusters/1 mm²3 clusters/1 mm²4 clusters/1 mm²5 clusters/1 mm²10 clusters/1 mm²50 clusters/1 mm²Or higher. In some cases, the substrate comprises about 1 tuft/10 mm²To about 10 clusters/1 mm². In some cases, the distance between the centers of two adjacent clusters is at least or about 50, 100, 200, 500, 1000, 2000, or 5000 um. In some cases, the distance between the centers of two adjacent clusters is about 50-100, 50-200, 50-300, 50-500, or 100-2000 um. In some cases, the distance between the centers of two adjacent clusters is about 0.05-50, 0.05-10, 0.05-5, 0.05-4, 0.05-3, 0.05-2, 0.1-10, 0.2-10, 0.3-10, 0.4-10, 0.5-5, or 0.5-2 mm. In some cases, each cluster independently has a cross-section of about 0.5 to 2, about 0.5 to 1, or about 1 to 2 mm. In some cases, each cluster independently has an average molecular weight of about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3,1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2mm cross-section. In some cases, each cluster independently has an internal cross-section of about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.15, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 mm.

In some cases, the substrate is about the size of a standard 96-well plate, e.g., about 100 to about 200mm by about 50 to about 150 mm. In some cases, the substrate has a diameter of less than or equal to about 1000, 500, 450, 400, 300, 250, 200, 150, 100, or 50 mm. In some cases, the diameter of the substrate is about 25-1000, 25-800, 25-600, 25-500, 25-400, 25-300, or 25-200 mm. In some cases, the substrate has at least about 100, 200, 500, 1,000, 2,000, 5,000, 10,000, 12,000, 15,000, 20,000, 30,000, 40,000, 50,000mm²Or a larger planar surface area. In some cases, the substrate has a thickness of about 50-2000, 50-1000, 100-1000, 200-1000, or 250-1000 mm.

Surfacing material

The substrates, devices, and reactors provided herein are made from any of a variety of materials suitable for the methods, compositions, and systems described herein. In some cases, the substrate material is fabricated to exhibit low levels of nucleotide incorporation. In some cases, the substrate material is modified to create different surfaces that exhibit high levels of nucleotide binding. In some cases, the substrate material is transparent to visible and/or ultraviolet light. In some cases, the substrate material is sufficiently conductive, e.g., capable of forming a uniform electric field across the entire substrate or a portion thereof. In some cases, the conductive material is electrically grounded. In some cases, the substrate is thermally conductive or thermally insulating. In some cases, the material is chemically and thermally resistant to support chemical or biochemical reactions, such as polynucleotide synthesis reaction processes. In some cases, the substrate comprises a flexible material. For flexible materials, the materials may include, but are not limited to: modified and unmodified nylon, nitrocellulose, polypropylene, and the like. In some cases, the substrate comprises a rigid material. For rigid materials, the materials may include, but are not limited to: glass; fused quartz; silicon, plastic (e.g., polytetrafluoroethylene, polypropylene, polystyrene, polycarbonate, mixtures thereof, and the like); metals (e.g., gold, platinum, etc.). The substrate, solid support or reactor may be made of a material selected from the group consisting of silicon, polystyrene, agarose, dextran, cellulose polymers, polyacrylamide, Polydimethylsiloxane (PDMS), and glass. The substrate/solid support or microstructures therein, the reactor, can be made using combinations of the materials listed herein or any other suitable material known in the art.

Surface structure

Provided herein are substrates for use in the methods, compositions, and systems described herein, wherein the substrates have a surface architecture suitable for the methods, compositions, and systems described herein. In some cases, the substrate comprises raised and/or recessed features. One benefit of having such features is the increased surface area available to support polynucleotide synthesis. In some cases, a substrate having raised and/or recessed features is referred to as a three-dimensional substrate. In some cases, the three-dimensional substrate comprises one or more channels. In some cases, one or more seats include a channel. In some cases, the channel may be subjected to reagent deposition by a deposition device such as a polynucleotide synthesizer. In some cases, reagents and/or fluids are collected in larger wells in fluid communication with one or more channels. For example, the substrate contains a plurality of channels corresponding to a plurality of seats within a cluster, and the plurality of channels are in fluid communication with one aperture of the cluster. In some methods, the polynucleotide library is synthesized in multiple loci of a cluster.

Provided herein are substrates for use in the methods, compositions, and systems described herein, wherein the substrates are configured for polynucleotide synthesis. In some cases, the structures are formulated to allow controlled flow and mass transfer pathways for polynucleotide synthesis on a surface. In some cases, the configuration of the substrate allows for controlled and uniform distribution of mass transfer paths, chemical exposure times, and/or wash efficacy during polynucleotide synthesis. In some cases, the configuration of the substrate allows for increased scanning efficiency, for example by providing a volume sufficient for growing the polynucleotide such that the volume excluded by the grown polynucleotide does not exceed 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the initial usable volume available for or suitable for growing the polynucleotide. In some cases, the three-dimensional structure allows for a managed flow of fluid, allowing for rapid exchange of chemical exposure.

Provided herein are substrates for use in methods, compositions, and systems related to the enzymatically mediated nucleic acid assembly and polynucleotide synthesis described herein, wherein the substrate comprises a structure configured to accommodate the enzymatic reactions described herein. In some cases, isolation is achieved by physical structures. In some cases, isolation is achieved by differential functionalization of the surface to generate activated and deactivated regions for polynucleotide synthesis. In some cases, differential functionalization is achieved by alternating hydrophobicity across the substrate surface, causing water contact angle effects that can cause beading or wetting of deposited reagents. The use of larger structures can reduce splatter and cross-contamination of different polynucleotide synthesis sites by reagents adjacent to the spots. In some cases, reagents are deposited at different polynucleotide synthesis locations using a device such as a polynucleotide synthesizer. Substrates with three-dimensional features are configured in a manner that allows for the synthesis of large numbers (e.g., more than about 10,000) of polynucleotides with low error rates (e.g., less than about 1:500, 1:1000, 1:1500, 1:2,000; 1:3,000; 1:5,000; or 1:10,000). In some cases, the substrate comprises a density of about or greater than about 1,5, 10, 20, 30, 40, 50, 60, 70, 80, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, or 500 features/mm²The characteristics of (1).

The aperture of the substrate may have the same or different width, height and/or volume as another aperture of the substrate. The channel of the substrate may have the same or different width, height, and/or volume as another channel of the substrate. In some cases, the diameter of the cluster or the diameter of the aperture containing the cluster or both is about 0.05-50, 0.05-10, 0.05-5, 0.05-4, 0.05-3, 0.05-2, 0.05-1, 0.05-0.5, 0.05-0.1, 0.1-10, 0.2-10, 0.3-10, 0.4-10, 0.5-5, or 0.5-2 mm. In some cases, the diameter of the tuft or hole, or both, is less than or about 5,4, 3, 2, 1, 0.5, 0.1, 0.09, 0.08, 0.07, 0.06, or 0.05 mm. In some cases, the diameter of the tuft or hole, or both, is about 1.0mm to about 1.3 mm. In some cases, the diameter of the tuft or hole, or both, is about 1.150 mm. In some cases, the diameter of the tuft or hole or both is about 0.08 mm. The diameter of a cluster refers to the cluster within a two-dimensional or three-dimensional substrate.

In some cases, the height of the wells is about 20-1000, 50-1000, 100-1000, 200-1000, 300-1000, 400-1000, or 500-1000. mu.m. In some cases, the height of the holes is less than about 1000, 900, 800, 700, or 600 um.

In some cases, the substrate comprises a plurality of channels corresponding to a plurality of loci within a cluster, wherein the height or depth of the channels is 5-500, 5-400, 5-300, 5-200, 5-100, 5-50, or 10-50 um. In some cases, the height of the channel is less than 100, 80, 60, 40, or 20 um.

In some cases, the diameter of the tunnel, the seat (e.g., in a substantially flat substrate), or both the tunnel and the seat (e.g., in a three-dimensional substrate in which the seat corresponds to the tunnel) is about 1-1000, 1-500, 1-200, 1-100, 5-100, or 10-100um, such as about 90, 80, 70, 60, 50, 40, 30, 20, or 10 um. In some cases, the diameter of the channel, the seat, or both the channel and the seat is less than about 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 um. In some cases, the distance between two adjacent channels, seats, or channels and the center of a seat is about 1-500, 1-200, 1-100, 5-200, 5-100, 5-50, or 5-30, for example about 20 um.

Surface modification

Provided herein are methods for synthesizing polynucleotides on a surface, wherein the surface comprises various surface modifications. In some cases, surface modification is used to chemically and/or physically alter the surface by an additive process or a subtractive process to alter one or more chemical and/or physical properties of the substrate surface or selected sites or regions of the substrate surface. For example, surface modifications include, but are not limited to: (1) changing the wetting properties of the surface; (2) functionalizing the surface, i.e., providing, modifying or replacing surface functional groups; (3) defunctionalizing the surface, i.e., removing surface functional groups; (4) changing the chemical composition of the surface in other ways, for example by etching; (5) increase or decrease surface roughness; (6) providing a coating on a surface, e.g., a coating that exhibits wetting properties that are different from the wetting properties of the surface; and/or (7) depositing particles on the surface.

In some cases, the addition of a chemical layer (referred to as an adhesion promoter) on top of the surface facilitates the structured patterning of the seats on the substrate surface. Exemplary surfaces for applying the adhesion promoter include, but are not limited to, glass, silicon dioxide, and silicon nitride. In some cases, the adhesion promoter is a chemical with high surface energy. In some cases, a second chemical layer is deposited on the surface of the substrate. In some cases, the second chemical layer has a low surface energy. In some cases, the surface energy of the chemical layer coated on the surface supports the positioning of droplets on the surface. Depending on the selected patterning arrangement, the proximity of the seats and/or the fluid contact area at the seats may be varied.

In some cases, the substrate surface or resolved loci onto which the nucleic acid or other moiety is deposited (e.g., for polynucleotide synthesis) are smooth or substantially planar (e.g., two-dimensional), or have irregularities such as raised or recessed features (e.g., three-dimensional features). In some cases, the substrate surface is modified with one or more different compound layers. Such modification layers of interest include, but are not limited to, inorganic and organic layers, such as metals, metal oxides, polymers, small organic molecules, and the like.

In some cases, the resolved loci of the substrate are functionalized with one or more moieties that increase and/or decrease surface energy. In some cases, the moiety is chemically inert. In some cases, the moiety is configured to support a desired chemical reaction, such as one or more processes in a polynucleotide synthesis reaction. The surface energy or hydrophobicity of a surface is a factor that determines the affinity of nucleotides for attaching to the surface. In some cases, a substrate functionalization method comprises: (a) providing a substrate having a surface comprising silicon dioxide; and (b) silanizing the surface using a suitable silylating agent (e.g., organofunctional alkoxysilane molecules) as described herein or known in the art. Methods and functionalizing agents are described in U.S. patent 5474796, which is incorporated herein by reference in its entirety.

In some cases, the substrate surface is functionalized, typically via reactive hydrophilic moieties present on the substrate surface, by contacting the substrate surface with a derivatizing composition comprising a mixture of silanes under reaction conditions effective to couple the silanes to the substrate surface. Silanization generally covers surfaces by self-assembly using organofunctional alkoxysilane molecules. A variety of siloxane functionalizing agents currently known in the art, for example, for reducing or increasing surface energy, may also be used. Organofunctional alkoxysilanes are classified according to their organofunctional group.

Computer system

Any of the systems described herein can be operatively connected to a computer and can be automated locally or remotely by the computer. In some cases, the methods and systems of the present invention further include software programs on a computer system and uses thereof. Thus, computerized control of the synchronization of the dispensing/vacuuming/refilling functions (e.g., programming and synchronizing material deposition device movement, dispensing action, and vacuum actuation) is within the scope of the present invention. The computer system can be programmed to interface between the user-specified base sequence and the location of the material deposition device to deliver the correct reagent to the specified region of the substrate.

The computer system 300 shown in fig. 3 may be understood as a logical device capable of reading instructions from media 311 and/or network port 305, which may optionally be connected to a server 309 having a fixed media 312. A system such as that shown in fig. 3 may include a CPU 301, a disk drive 303, optional input devices such as a keyboard 315 and/or mouse 316, and an optional monitor 307. Data communication with a server at a local or remote location may be accomplished through the communication media shown. A communication medium may include any means for transmitting and/or receiving data. The communication medium may be a network connection, a wireless connection, or an internet connection, for example. Such connections may provide for communication via the world wide web. It is contemplated that data related to the present disclosure may be transmitted over such a network or connection for receipt and/or review by user party 322 as shown in fig. 3.

FIG. 4 is a block diagram illustrating an architecture of a computer system 400 that may be used in connection with the exemplary embodiments of this invention. As shown in FIG. 4, the example computer system may include a processor 402 for processing instructions. Non-limiting examples of processors include:processor, AMD Opteron^TMA processor, a Samsung 32-bit RISC ARM 1176JZ (F) -S v 1.0.0 processor, an ARM Cortex-A8 Samsung S5PC100 processor, an ARM Cortex-A8 Apple A4 processor, a Marvell PXA 930 processor, or a functionally equivalent processor. Multiple threads of execution may be used for parallel processing. In some cases, multiple processors or processors with multiple cores may also be used, whether in a single computer system, in a cluster, or distributed across a system by a network containing multiple computers, cell phones, and/or personal data assistant devices.

As shown in FIG. 4, a cache memory 404 may be connected to or incorporated into the processor 402 to provide a high-speed store of instructions or data that are recently or frequently used by the processor 402. Processor 402 is coupled to north bridge 406 by processor bus 408. Northbridge 406 connects to Random Access Memory (RAM)410 through memory bus 412, and manages access to RAM410 by processor 402. The north bridge 406 is also connected to a south bridge 414 through a chipset bus 416. South bridge 414, in turn, is connected to peripheral bus 418. The peripheral bus may be, for example, PCI-X, PCI Express, or other peripheral bus. The north bridge and south bridge are commonly referred to as a processor chipset and manage data transfers between the processor, RAM, and peripheral components on the peripheral bus 418. In some alternative architectures, the functionality of the north bridge may be incorporated into the processor, rather than using a separate north bridge chip. In some cases, system 400 may include an accelerator card 422 attached to peripheral bus 418. The accelerator may include a Field Programmable Gate Array (FPGA) or other hardware for accelerating some processing. For example, the accelerator may be used for adaptive data reconstruction or to evaluate algebraic expressions used in extended set processing.

Software and data are stored in the external memory 424 and may be loaded into the RAM410 and/or the cache 404 for use by the processor. System 400 includes an operating system for managing system resources; non-limiting examples of operating systems include: linux, windows, MACOSTM, BlackBerry OSTM, iOSTM, and other functionally equivalent operating systems, and application software running on top of the operating systems for managing data storage and optimization according to example embodiments of the present invention. In this example, system 400 also includes Network Interface Cards (NICs) 420 and 421 connected to the peripheral bus to provide a network interface to external storage, such as Network Attached Storage (NAS) and other computer systems available for distributed parallel processing.

FIG. 5 is a block diagram of a multiprocessor computer system using a shared virtual address memory space according to an example embodiment. The system includes a plurality of processors 502a-f that may access a shared memory subsystem 504. In this system a plurality of programmable hardware storage algorithm processors (MAPs) 506a-f are incorporated in a memory subsystem 504. Each MAP 506a-f may contain a memory 508a-f and one or more Field Programmable Gate Arrays (FPGAs) 510 a-f. The MAP provides configurable functional units and may provide specific algorithms or portions of algorithms to the FPGAs 510a-f for processing in close cooperation with the respective processors. For example, in an exemplary embodiment, MAP may be used to evaluate algebraic expressions associated with data models and to perform adaptive data reconstruction. In this example, each MAP is globally accessible by all processors for these purposes. In one configuration, each MAP may use Direct Memory Access (DMA) to access the associated memory 508a-f to perform tasks independently and asynchronously from the respective microprocessor 502 a-f. In this configuration, a MAP may feed the results directly to another MAP for pipelined processing and parallel execution of algorithms.

FIG. 6 is a diagram showing a network having multiple computer systems 602a and 602b, multiple cellular telephones and personal data assistants 602c, and Network Attached Storage (NAS)604a and 604 b. In an example embodiment, the systems 602a, 602b, and 602c may manage data storage and optimize data access to data stored in Network Attached Storage (NAS)604a and 604 b. A mathematical model may be used for this data and evaluated using distributed parallel processing across computer systems 602a and 602b and cellular phones and personal data assistant system 602 c. Computer systems 602a and 602b and cellular telephone and personal data assistant system 602c may also provide parallel processing of adaptive data reconstruction of data stored in Network Attached Storage (NAS)604a and 604 b. FIG. 6 illustrates only one example, and a wide variety of other computer architectures and systems can be used with embodiments of the present invention. For example, blade servers may be used to provide parallel processing. Processor blades may be connected through a backplane to provide parallel processing. The storage may also be connected to the backplane through a separate network interface or as Network Attached Storage (NAS). In some examples, the processors may maintain separate memory spaces and transmit data through a network interface, backplane, or other connector for parallel processing by other processors. In some cases, some or all of the processors may use a shared virtual address memory space.

Any of the systems described herein can include sequence information stored on a non-transitory computer readable storage medium. In some cases, any of the systems described herein include a computer input file. In some cases, the computer input file contains sequence information. In some cases, the computer input file comprises instructions for synthesizing a plurality of polynucleotide sequences. In some cases, the instructions are received by a computer. In some cases, the instruction is processed by the computer. In some cases, the instructions are communicated to a material deposition device. In some cases, the non-transitory computer readable storage medium is encoded with a program comprising instructions executable by an operating system of an optionally networked digital processing device. In some cases, the computer readable storage medium is a tangible component of a digital processing device. In some cases, the computer readable storage medium is optionally removable from the digital processing apparatus. In some cases, computer-readable storage media include CD-ROMs, DVDs, flash memory devices, solid state memory, magnetic disk drives, tape drives, optical disk drives, cloud computing systems and services, and the like, to name a non-limiting example. In some cases, programs and instructions are encoded on media permanently, substantially permanently, semi-permanently, or non-temporarily.