NUCLEIC ACID AMPLIFICATION AND SEQUENCING BY SYNTHESIS WITH FLUOROGENIC NUCLEOTIDES

• US 20130053252A1
• Filed: 09/24/2010
• Published: 02/28/2013
• Est. Priority Date: 09/25/2009
• Status: Abandoned Application

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Xie, Xiaoliang Sunney, Sims, Peter A., Greenleaf, William J., Duan, Haifeng
• Application Number:
  US13/498,072
• Publication Number:
  US 20130053252A1
• Time in Patent Office: Days
• US Class Current: 506/2
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation

Abstract

In general, the invention features methods and systems for sequencing of nucleic acids based on the measurement of the incorporation of fluorogenic nucleotides in microreactors. The invention provides numerous advantages over previous systems such as unambiguous determination of sequence, fast cycle time, long read lengths, low overall cost of reagents, low instrument cost, and high throughput. The invention also features methods and kits for nucleic acid amplification. The amplification and sequencing aspects of the invention may or may not be employed in conjunction with one another. The invention also features fluorogenic nucleotides that may be used in the sequencing methods of the invention.

Claims

1. A method for sequencing a nucleic acid, said method comprising the steps of:

a) immobilizing in an optionally sealed microreactor a single target nucleic acid or a plurality of copies of the target nucleic acid;

b) introducing to the microreactor a mixture in solution phase comprising a nucleic acid replicating catalyst, and a single species of nucleotide comprising a first base and a first label that is substantially non-fluorescent until after incorporation of said nucleotide into a nucleic acid based on complementarity to said target nucleic acid;

c) allowing template-dependent replication of said target nucleic acid or the plurality of copies of said target nucleic acid; and

d) sequencing said target nucleic acid by detecting incorporation of said nucleotide during template-dependent replication by detecting fluorescence emission resulting from said first label.

2. The method of claim 1, wherein said mixture in solution phase further comprises an activating enzyme that renders said first label fluorescent.

7. The method of claim 1, wherein said mixture in solution phase further comprises non-hydrolyzable nucleotides that compete for binding to the nucleic acid replicating catalyst to prevent misincorporation of the nucleotide.

8. The method of claim 1, wherein, subsequent to step (d), a second mixture in solution phase comprising an unlabeled nucleotide species comprising the first base is introduced into the microreactor and template-dependent replication is allowed to proceed until the sequencing cycle is complete.

10. The method of claim 1, wherein steps (b)-(d) are repeated with a second single nucleotide species comprising a second base and a second label that is substantially non-fluorescent until incorporation of said second nucleotide into said nucleic acid based on complementarity to said target nucleic acid, wherein the first and second labels are the same or different, and the first and second bases are different.

14. The method of claim 1, wherein the microreactor is sealed.

18. The method of claim 1, wherein said nucleic acid replicating catalyst is a DNA polymerase, RNA polymerase, ligase, reverse transcriptase, or RNA-dependent RNA polymerase.

22. The method of claim 1, wherein said target nucleic acid or plurality of copies is immobilized on a bead disposed in said microreactor.

23. The method of claim 1, wherein said plurality of copies is immobilized in step (a).

32. The method of claim 1, wherein the single species of nucleotide further comprises a reversible terminator.

35. The method of claim 1, wherein the mixture in solution phase further comprises an exonuclease, wherein a plurality of first labels are produced as a result of incorporation of the nucleotide and subsequent excision by the exonuclease.

39. The method of claim 1, further comprising, prior to step (a), introducing said target nucleic acid, which is reversibly bound to a bead, into said microreactor.

40. The method of claim 1, wherein, in step (a), (i) said microreactor comprises bound oligonucleotides, (ii) a nucleic acid complementary to said target nucleic acid and reversibly bound to a bead is introduced into said microreactor, wherein said complementary nucleic acid binds to one of said bound oligonucleotides, and (iii) said bound oligonucleotide is extended via template-dependent replication, thereby immobilizing said target nucleic acid in said microreactor.

43. The method of claim 1, wherein, prior to step (b), the microreactor is cooled to 15°

C. or lower.

45. The method of claim 1, further comprising a population of single target nucleic acids or a population of pluralities of copies of the target nucleic acids, wherein each single target nucleic acid or plurality of copies of the target nucleic acid is immobilized in one of a plurality of microreactors, and steps (b)-(d) are performed for the population.

3. (canceled)

4. (canceled)

5. (canceled)

6. (canceled)

9. (canceled)

11. (canceled)

12. (canceled)

13. (canceled)

15. (canceled)

16. (canceled)

17. (canceled)

19. (canceled)

20. (canceled)

21. (canceled)

24. (canceled)

25. (canceled)

26. (canceled)

27. (canceled)

28. (canceled)

29. (canceled)

30. (canceled)

31. (canceled)

33. (canceled)

34. (canceled)

36. (canceled)

37. (canceled)

38. (canceled)

41. (canceled)

42. (canceled)

44. (canceled)

46. (canceled)

47. (canceled)

48. (canceled)

49. (canceled)

50. (canceled)

51. (canceled)

52. (canceled)

53. (canceled)

54. (canceled)

55. (canceled)

56. (canceled)

57. (canceled)

58. A method for sequencing a nucleic acid, said method comprising the steps of:

a) immobilizing in a microreactor a single target nucleic acid or a plurality of copies of the target nucleic acid;

b) cooling said microreactor to 15°

C. or lower;

c) introducing to the microreactor a mixture in solution phase comprising a nucleic acid replicating catalyst, and a single species of nucleotide comprising a first base and a first label that is substantially non-fluorescent until after incorporation of said nucleotide into a nucleic acid based on complementarity to said target nucleic acid;

d) sealing said microreactor and heating said microreactor to 20°

C. or higher;

e) allowing template-dependent replication of said target nucleic acid or the plurality of copies of said target nucleic acid;

f) sequencing said target nucleic acid by detecting incorporation of said nucleotide during template-dependent replication by detecting fluorescence emission resulting from said first label;

g) repeating steps b)-f) sequentially with a second single nucleotide species comprising a second base and a second label that is substantially non-fluorescent until incorporation of said second nucleotide into said nucleic acid based on complementarity to said target nucleic acid, a third single nucleotide species comprising a third base and a third label that is substantially non-fluorescent until incorporation of said third nucleotide into said nucleic acid based on complementarity to said target nucleic acid; and

a fourth single nucleotide species comprising a fourth base and a fourth label that is substantially non-fluorescent until incorporation of said fourth nucleotide into said nucleic acid based on complementarity to said target nucleic acid, wherein any two of the first, second, third and fourth labels are the same or different, and the first, second, third, and fourth bases are different.

59. A method of amplifying a nucleic acid, said method comprising the steps of:

a) providing a single copy of a first nucleic acid having first and second ends;

b) immobilizing the first nucleic acid via the first end to a bead;

c) immobilizing the second end of the nucleic acid to a surface of a microreactor; and

d) amplifying the first nucleic acid to produce a plurality of amplicons having first and second ends, wherein the plurality of amplicons binds to the surface of the microreactor via the second ends or to the bead via the first ends;

or

a) providing a single copy of a first nucleic acid having first and second ends;

b) immobilizing the second end of the nucleic acid to a surface of a microreactor; and

c) amplifying the first nucleic acid to produce a plurality of amplicons having first and second ends, wherein the plurality of amplicons binds to the surface of the microreactor via the second ends;

or

a) providing a single copy of a first nucleic acid having first and second ends;

b) optionally immobilizing the first nucleic acid via the first end to a bead;

c) immobilizing the second end of the first nucleic acid to one of a plurality of complementary oligonucleotides bound to a surface of a microreactor;

d) extending the oligonucleotide by template dependent replication to produce a second nucleic acid bound to the surface of the microreactor; and

e) amplifying the second nucleic acid to produce a plurality of amplicons extended from said plurality of oligonucleotides bound to said surface of said microreactor;

or

a) providing a single copy of a first circular nucleic acid;

b) immobilizing the first nucleic acid to one of a plurality of complementary oligonucleotides bound to a surface of a microreactor or a bead;

c) extending the oligonucleotide by rolling circle amplification to produce a second nucleic acid bound to the surface of the microreactor or bead; and

d) amplifying the second nucleic acid to produce a plurality of amplicons extended from said plurality of oligonucleotides bound to said surface of said microreactor.

60. (canceled)

61. (canceled)

62. (canceled)

63. (canceled)

64. (canceled)

65. (canceled)

66. (canceled)

67. (canceled)

68. (canceled)

69. (canceled)

70. (canceled)

71. (canceled)

72. (canceled)

73. (canceled)

74. (canceled)

75. (canceled)

76. (canceled)

77. (canceled)

78. (canceled)

79. (canceled)

80. (canceled)

81. (canceled)

82. (canceled)

83. (canceled)

84. (canceled)

85. (canceled)

86. (canceled)

87. (canceled)

88. (canceled)

89. (canceled)

90. (canceled)

91. (canceled)

92. (canceled)

93. (canceled)

94. (canceled)

95. (canceled)

96. (canceled)

97. A system for sequencing a nucleic acid comprising:

a plurality of microreactors that are each capable of holding an immobilized single target nucleic acid or plurality of copies of said target nucleic acid, a mixture in solution phase of a nucleic acid replicating catalyst, and a single species of nucleotide that comprises a label that is substantially non-fluorescent until after incorporation of at least one nucleotide into a nucleic acid based on complementarity to said target nucleic acid;

a fluorescent microscope for imaging said plurality of microreactors to sequence target nucleic acids in said microreactors by detecting in each microreactor the incorporation of an individual nucleotide species during template-dependent replication of said single copy of said target nucleic acid by monitoring fluorescence from said labels resulting from incorporation of said at least one nucleotide; and

a fluidic delivery system capable of delivering liquids from each of four reservoirs to each of said plurality of microreactors.

98. (canceled)

99. (canceled)

100. (canceled)

101. (canceled)

102. (canceled)

103. (canceled)

104. (canceled)

105. (canceled)

106. (canceled)

107. (canceled)

108. (canceled)

109. (canceled)

110. (canceled)

111. (canceled)

112. (canceled)

113. A compound selected from the group consisting of formula:

114. (canceled)

115. A kit comprising:

a plurality of microreactors that are each capable of holding an immobilized single target nucleic acid, a mixture in solution phase of reagents for template dependent replication of the single target nucleic acid, and a bead functionalized to bind to the single target nucleic acid;

a plurality of beads that are each capable of binding a nucleic acid and being disposed within one of the microreactors; and

reagents for template dependent replication of the nucleic acid.

116. (canceled)

117. (canceled)

118. (canceled)

119. (canceled)