2'-terminator nucleotide-related methods and systems

• US 20050037398A1
• Filed: 06/28/2004
• Published: 02/17/2005
• Est. Priority Date: 06/30/2003
• Status: Active Grant

• Current Assignee: Roche Molecular Systems (Roche Holding AG)
• Original Assignee: Roche Molecular Systems (Roche Holding AG)
• Inventors: Bodepudi, Veeraiah, Gupta, Amar, Myers, Thomas, Will, Stephen, Reichert, Fred Lawrence, Gelfand, David Harrow
• Granted Patent: US 7,572,581 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• CPC Class Codes: Y10S 435/975 Kit

Abstract

The present invention provides methods of extending primer nucleic acids and sequencing target nucleic acids. The methods include the use of 2′-terminator nucleotides to effect chain termination. In addition to related reaction mixtures and kits, the invention also provides computers and computer readable media.

Claims

1. A method of extending a primer nucleic acid, the method comprising:

incubating a template nucleic acid with at least one nucleotide incorporating biocatalyst, at least one 2′

-terminator nucleotide, and at least one primer nucleic acid that is at least partially complementary to at least a subsequence of the template nucleic acid, which primer nucleic acid comprises DNA, under conditions whereby the nucleotide incorporating biocatalyst extends the primer nucleic acid to produce at least one extended primer nucleic acid by incorporating the 2′

-terminator nucleotide at a terminal end of the extended primer nucleic acid.

2. The method of claim 1, wherein the template nucleic acid comprises DNA.

3. The method of claim 1, wherein the template nucleic acid comprises RNA.

4. The method of claim 1, wherein the 2′

-terminator nucleotide comprises a 2′

-monophosphate-3′

-hydroxyl-5′

-triphosphate nucleoside or a negatively charged blocking group.

5. The method of claim 1, wherein a 2′

position of a sugar moiety of the 2′

-terminator nucleotide comprises a bulky blocking group.

6. The method of claim 1, wherein the terminal end of the extended primer nucleic acid is the 3′

terminal end.

7. The method of claim 1, wherein the nucleotide incorporating biocatalyst comprises an enzyme selected from the group consisting of:

a polymerase, a terminal transferase, a reverse transcriptase, a polynucleotide phosphorylase, and a telomerase.

8. The method of claim 7, wherein the enzyme comprises a 3′

-5′

exonuclease activity.

9. The method of claim 7, wherein the enzyme comprises a mutation that enhances incorporation of ribonucleotides.

10. The method of claim 7, wherein the enzyme comprises a mutation that reduces or eliminates 5′

-3′

exonuclease activity.

11. The method of claim 7, wherein the enzyme comprises a mutation that enhances incorporation of 2′

-modified analogs of ribonucleotides.

12. The method of claim 7, wherein the enzyme comprises a thermostable enzyme.

13. The method of claim 7, wherein the enzyme is derived from an organism that is selected from the group consisting of:

Thermus antranikianii, Thermus aquaticus, Thermus caldophilus, Thermus chliarophilus, Thermus filiformis, Thermus flavus, Thermus igniterrae, Thermus lacteus, Thermus oshimai, Thermus ruber, Thermus rubens, Thermus scotoductus, Thermus silvanus, Thermus species Z05, Thermus species sps 17, Thermus thermophilus, Thermotoga maritima, Thermotoga neapolitana, Thermosipho africanus, Anaerocellum thermophilum, Bacillus caldotenax, and Bacillus stearothermophilus.

14. The method of claim 7, wherein the enzyme is modified.

15. The method of claim 14, wherein the modified enzyme comprises one or more of:

a G46E E678G CS5 DNA polymerase, a G46E E678G CS6 DNA polymerase, an E615G Taq DNA polymerase,a Δ

ZO5R polymerase, or a G46E L329A E678G CS5 DNA polymerase.

16. The method of claim 1, further comprising detecting all or a portion of the extended primer nucleic acid or a fragment thereof.

17. The method of claim 16, wherein the extended primer nucleic acid or the fragment thereof is detected using gas phase ion spectrometry.

18. The method of claim 17, wherein the gas phase ion spectrometry comprises MALDI-TOF mass spectrometry.

19. The method of claim 1, wherein at least one of the 2′

-terminator nucleotide, the extended primer nucleic acid, and the primer nucleic acid comprises at least one label.

20. The method of claim 19, wherein a label is attached to one of a heterocyclic base of the 2′

-terminator nucleotide, a sugar moiety of the 2′

-terminator nucleotide, and a phosphate group of the 2′

-terminator nucleotide.

21. The method of claim 19, wherein a linker attaches a label to the 2′

-terminator nucleotide.

22. The method of claim 19, further comprising detecting a detectable signal produced by the label.

23. The method of claim 19, wherein the label comprises a fluorescent dye, a weakly fluorescent label, a non-fluorescent label, a colorimetric label, a chemiluminescent label, a bioluminescent label, a radioisotope, an antibody, an antigen, biotin, a hapten, or an enzyme.

24. The method of claim 23, wherein the label is a fluorescent dye selected from the group consisting of:

a rhodamine dye, a fluorescein dye, a halofluorescein dye, a dichlororhodamine dye, an energy transfer dye, a Lucifer dye, Oregon Green, and a cyanine dye.

25. The method of claim 23, wherein the label is a fluorescent dye selected from the group consisting of:

JOE, VIC, TET, HEX, FAM, R6G, R110, TAMRA, and ROX.

26. The method of claim 23, wherein the label is a radioisotope selected from the group consisting of:

³H, ¹⁴C, ²²Na, ³²P, ³³P, ³⁵S, ⁴²K, ⁴⁵Ca, ⁵⁹Fe, ¹²⁵I, and ²⁰³Hg.

27. The method of claim 1, wherein the template nucleic acid or the primer nucleic acid is attached to a solid support.

28. The method of claim 1, further comprising incubating the template nucleic acid with at least one extendible nucleotide.

29. The method of claim 28, wherein the extendible nucleotide comprises a ribonucleotide and/or a deoxyribonucleotide.

30. The method of claim 29, wherein the extendible nucleotide is labeled.

31. The method of claim 28, comprising incubating the template nucleic acid with at least one pyrophosphatase that minimizes pyrophosphorolysis.

32. The method of claim 28, wherein the 2′

-terminator nucleotides and the extendible nucleotides are present in a molar ratio of 1;

1 or less.

33. The method of claim 28, wherein the extended primer nucleic acid is complementary to a subsequence of the template nucleic acid.

34. The method of claim 28, wherein the extended primer nucleic acid is complementary to a full-length sequence of the template nucleic acid.

35. The method of claim 28, wherein the nucleotide incorporating biocatalyst produces multiple different extended primer nucleic acids and the method comprises resolving the multiple different extended primer nucleic acids, whereby at least a portion of a base sequence of the template nucleic acid is determinable from the resolved extended primer nucleic acids.

36. The method of claim 35, wherein the extended primer nucleic acids are resolved by determining at least one of the molecular masses, sizes, and charge properties of the extended primer nucleic acids.

37. The method of claim 35, wherein the extended primer nucleic acids further comprise labels and the extended primer nucleic acids are resolved by separating the labeled extended primer nucleic acids from each other and detecting detectable signals produced by the labels.

38. The method of claim 37, wherein the labeled extended primer nucleic acids are separated by at least one separation technique selected from the group consisting of electrophoresis, chromatography, and gas phase ion spectrometry.

39. The method of claim 38, wherein the gas phase ion spectrometry comprises MALDI-TOF mass spectrometry.

40. A method of extending a nucleic acid, the method comprising:

incubating at least one nucleic acid with at least one nucleotide incorporating biocatalyst and at least one labeled 2′

-terminator nucleotide, whereby the nucleotide incorporating biocatalyst extends the nucleic acid to produce at least one extended nucleic acid by incorporating the labeled 2′

-terminator nucleotide at a terminal end of the nucleic acid.

41. The method of claim 40, wherein the nucleotide incorporating biocatalyst comprises an enzyme selected from the group consisting of:

a terminal transferase and a polynucleotide phosphorylase.

42. The method of claim 40, wherein the labeled 2′

-terminator nucleotide comprises a 2′

-monophosphate-3′

-hydroxyl-5′

-triphosphate nucleoside.

43. The method of claim 40, wherein the nucleotide incorporating biocatalyst incorporates the labeled 2′

-terminator nucleotide at a 3′

terminal end of the nucleic acid.

44. The method of claim 40, further comprising hybridizing the extended nucleic acid with another nucleic acid and detecting a detectable signal produced by the label.

45. The method of claim 40, wherein the label comprises a fluorescent dye.

46. The method of claim 40, wherein the label comprises a radioisotope.

47. The method of claim 40, wherein the label comprises at least one mass-modifying group.

48. The method of claim 40, wherein the nucleic acid comprises a primer nucleic acid that is at least partially complementary to at least a subsequence of a template nucleic acid, and the method comprises incubating the template nucleic acid with the nucleotide incorporating biocatalyst, the labeled 2′

-terminator nucleotide, and the primer nucleic acid.

49. The method of claim 48, wherein the nucleotide incorporating biocatalyst comprises an enzyme selected from the group consisting of:

a polymerase, a terminal transferase, a reverse transcriptase, a polynucleotide phosphorylase, and a telomerase.

50. The method of claim 48, wherein the nucleotide incorporating biocatalyst comprises a modified enzyme.

51. The method of claim 50, wherein the modified enzyme comprises one or more of:

a G46E E678G CS5 DNA polymerase, a G46E E678G CS6 DNA polymerase, an E615G Taq DNA polymerase, a Δ

ZO5R polymerase, or a G46E L329A E678G CS5 DNA polymerase.

52. The method of claim 48, further comprising incubating the template nucleic acid with at least one extendible nucleotide.

53. The method of claim 52, wherein the nucleotide incorporating biocatalyst produces multiple different extended primer nucleic acids and the method comprises resolving the multiple different extended primer nucleic acids, whereby at least a portion of a base sequence of the template nucleic acid is determinable from the resolved extended primer nucleic acids.

54. The method of claim 53, wherein the extended primer nucleic acids are resolved by determining at least one of the molecular masses, sizes, and charge properties of the extended primer nucleic acids.

55. The method of claim 53, wherein the extended primer nucleic acids are resolved by separating the extended primer nucleic acids from each other and detecting detectable signals produced by the labels.

56. A method of inhibiting further extension of an extended nucleic acid, the method comprising:

contacting at least one nucleic acid with at least one nucleotide incorporating biocatalyst and at least one 2′

-terminator nucleoside or nucleotide, or a pharmaceutically acceptable salt thereof, which nucleic acid comprises DNA and which 2′

-terminator nucleoside or nucleotide, or pharmaceutically acceptable salt thereof is non-extendible by the nucleotide incorporating biocatalyst, whereby the nucleotide incorporating biocatalyst extends the nucleic acid to produce at least one extended nucleic acid by incorporating the labeled 2′

-terminator nucleoside or nucleotide, or the pharmaceutically acceptable salt thereof, at a terminal end of the nucleic acid, thereby inhibiting further extension of the extended nucleic acid.

57. The method of claim 56, wherein the nucleic acid comprises microbial DNA.

58. The method of claim 57, wherein the microbial DNA, the nucleotide incorporating biocatalyst, and the 2′

-terminator nucleoside or nucleotide, or the pharmaceutically acceptable salt thereof, are contacted in a host infected with a microbe that comprises the microbial DNA.

59. A method of sequencing a target nucleic acid, the method comprising:

(a) incubating at least one target nucleic acid with one or more polymerases, one or more 2′

-monophosphate-3′

-hydroxyl nucleosides, one or more extendible nucleotides, and one or more primer nucleic acids that are complementary to at least a subsequence of the target nucleic acid, whereby the polymerases extend the primer nucleic acids to produce primer extension products that incorporate the 2′

-monophosphate-3′

-hydroxyl nucleosides at 3′

-terminal ends of the primer extension products; and

,

(b) identifying the 2′

-monophosphate-3′

-hydroxyl nucleosides in the primer extension products, whereby at least a portion of a base sequence of the target nucleic acid is determinable from the identified 2′

-monophosphate-3′

-hydroxyl nucleosides.

60. The method of claim 59, wherein (a) comprises incubating the target nucleic acid, the polymerases, the extendible nucleotides, and the primer nucleic acids with at least two different 2′

-monophosphate-3′

-hydroxyl nucleosides.

61. The method of claim 59, wherein (a) comprises multiple separate reactions, wherein at least two of the reactions comprise different 2′

-monophosphate-3′

-hydroxyl nucleosides.

62. The method of claim 61, wherein the different 2′

-monophosphate-3′

-hydroxyl nucleosides comprise different labels.

63. The method of claim 59, wherein (b) comprises determining the molecular masses of the primer extension products or 3′

-terminal fragments thereof and the sequence of the target nucleic acid from the molecular masses.

64. The method of claim 63, wherein the molecular masses are determined using gas phase ion spectrometry.

65. The method of claim 64, wherein the gas phase ion spectrometry comprises MALDI-TOF mass spectrometry.

66. The method of claim 59, wherein the primer extension products comprise labels and (b) comprises separating the primer extension products from each other and detecting detectable signals produced by the labels.

67. The method of claim 66, wherein the primer extension products are separated by one or more separation techniques selected from the group consisting of electrophoresis, chromatography, and gas phase ion spectrometry.

68. A reaction mixture comprising at least one labeled 2′

-terminator nucleotide and at least one nucleotide incorporating biocatalyst.

69. The reaction mixture of claim 68, wherein a 2′

position of a sugar moiety of the 2′

-terminator nucleotide comprises a negatively charged blocking group or a bulky blocking group.

70. The reaction mixture of claim 68, wherein the 2′

-terminator nucleotide comprises a 2′

-monophosphate-3′

-hydroxyl nucleoside.

71. The reaction mixture of claim 68, further comprising at least one pyrophosphatase.

72. The reaction mixture of claim 68, wherein the 2′

-terminator nucleotide comprises the formula;

73. The reaction mixture of claim 72, wherein B comprises a formula selected from the group consisting of:

74. The reaction mixture of claim 72, wherein BG is selected from the group consisting of:

CN, NO₂, N₃, a silyl group, a halo group, an alcohol group, an ether group, an aldehyde group, an acidic group, an ester group, an amino group, and combinations thereof.

75. The reaction mixture of claim 72, wherein BG comprises the formula:

76. The reaction mixture of claim 68, wherein the label is attached to one of a heterocyclic base of the 2′

-terminator nucleotide, a sugar moiety of the 2′

-terminator nucleotide, and a phosphate group of the 2′

-terminator nucleotide.

77. The reaction mixture of claim 68, wherein a linker attaches the label to the 2′

-terminator nucleotide.

78. The reaction mixture of claim 68, wherein the label comprises a fluorescent dye, a weakly fluorescent label, a non-fluorescent label, a colorimetric label, a chemiluminescent label, a bioluminescent label, a radioisotope, an antibody, an antigen, biotin, a hapten, or an enzyme.

79. The reaction mixture of claim 78, wherein the label is a fluorescent dye selected from the group consisting of:

a rhodamine dye, a fluorescein dye, a halofluorescein dye, a dichlororhodamine dye, an energy transfer dye, a Lucifer dye, Oregon Green, and a cyanine dye.

80. The reaction mixture of claim 79, wherein the fluorescent dye is selected from the group consisting of:

JOE, VIC, TET, HEX, FAM, R6G, R110, TAMRA, and ROX.

81. The reaction mixture of claim 78, wherein the label is a radioisotope selected from the group consisting of:

³H, ¹⁴C, ²²Na, ³²P, ³³P, ³⁵S, ⁴²K, ⁴⁵Ca, ⁵⁹Fe, ¹²⁵I, and ²⁰³Hg.

82. The reaction mixture of claim 68, further comprising one or more extendible nucleotides.

83. The reaction mixture of claim 82, wherein the extendible nucleotides comprise ribonucleotides and/or deoxyribonucleotides.

84. The reaction mixture of claim 82, wherein at least one of the extendible nucleotides is labeled.

85. The reaction mixture of claim 82, wherein the 2′

-terminator nucleotide and the extendible nucleotides are present in a molar ratio of 1;

1 or less.

86. The reaction mixture of claim 68, wherein the nucleotide incorporating biocatalyst comprises an enzyme selected from the group consisting of:

a polymerase, a terminal transferase, a reverse transcriptase, a polynucleotide phosphorylase, and a telomerase.

87. The reaction mixture of claim 86, wherein the enzyme comprises a 3′

-5′

exonuclease activity.

88. The reaction mixture of claim 86, wherein the enzyme comprises a mutation that enhances incorporation of ribonucleotides.

89. The reaction mixture of claim 86, wherein the enzyme comprises a mutation that reduces or eliminates 5′

-3′

exonuclease activity.

90. The reaction mixture of claim 86, wherein the enzyme comprises a mutation that enhances incorporation of 2′

-modified analogs of ribonucleotides.

91. The reaction mixture of claim 86, wherein the enzyme comprises a thermostable enzyme.

92. The reaction mixture of claim 86, wherein the enzyme is derived from an organism that is selected from the group consisting of:

Thermus antranikianii, Thermus aquaticus, Thermus caldophilus, Thermus chliarophilus, Thermus filiformis, Thermus flavus, Thermus igniterrae, Thermus lacteus, Thermus oshimai, Thermus ruber, Thermus rubens, Thermus scotoductus, Thermus silvanus, Thermus species Z05, Thermus species sps 17, Thermus thermophilus, Thermotoga maritima, Thermotoga neapolitana, Thermosipho africanus, Anaerocellum thermophilum, Bacillus caldotenax, and Bacillus stearothermophilus.

93. The reaction mixture of claim 86, wherein the enzyme is modified.

94. The reaction mixture of claim 93, wherein the modified enzyme comprises one or more of:

is a G46E E678G CS5 DNA polymerase, a G46E E678G CS6 DNA polymerase, an E615G Taq DNA polymerase, a Δ

ZO5R polymerase, or a G46E L329A E678G CS5 DNA polymerase.

95. The reaction mixture of claim 68, further comprising a template nucleic acid and a primer nucleic acid that is at least partially complementary to at least a subsequence of the template nucleic acid.

96. The reaction mixture of claim 95, wherein the template nucleic acid or the primer nucleic acid is attached to a solid support.

97. The reaction mixture of claim 95, wherein the primer comprises a label.

98. The reaction mixture of claim 97, wherein the label comprises a fluorescent dye, a weakly fluorescent label, a non-fluorescent label, a colorimetric label, a chemiluminescent label, a bioluminescent label, a radioisotope, an antibody, an antigen, biotin, a hapten, or an enzyme.

99. The reaction mixture of claim 98, wherein label is a fluorescent dye selected from the group consisting of:

a rhodamine dye, a fluorescein dye, a halofluorescein dye, a dichlororhodamine dye, an energy transfer dye, a Lucifer dye, Oregon Green, and a cyanine dye.

100. The reaction mixture of claim 99, wherein the fluorescent dye is selected from the group consisting of:

JOE, VIC, TET, HEX, FAM, R6G, R110, TAMRA, and ROX.

101. The reaction mixture of claim 97, wherein the label comprises a radioisotope selected from the group consisting of:

³H, ¹⁴C, ²²Na, ³²P, ³³P, ³⁵S, ⁴²K, ⁴⁵Ca, ⁵⁹Fe, ¹²⁵I, and ²⁰³Hg.

102. A kit for extending a nucleic acid, comprising:

(a) at least one nucleotide incorporating biocatalyst; and

,

(b) at least one labeled 2′

-terminator nucleotide.

103. The kit of claim 102, wherein a 2′

position of a sugar moiety of the labeled 2′

-terminator nucleotide comprises a negatively charged blocking group or a bulky blocking group.

104. The kit of claim 102, wherein the 2′

-terminator nucleotide comprises a 2′

-monophosphate-3′

-hydroxyl nucleoside.

105. The kit of claim 102, further comprising one or more extendible nucleotides.

106. The kit of claim 105, wherein at least one of the extendible nucleotides comprises a label.

107. The kit of claim 106, wherein the label comprises a radioisotope, a fluorescent dye, or a mass-modifying group.

108. The kit of claim 102, further comprising at least one pyrophosphatase.

109. The kit of claim 102, further comprising:

(c) a set of instructions for extending the nucleic acid with the nucleotide incorporating biocatalyst and the labeled 2′

-terminator nucleotide.

110. The kit of claim 109, further comprising:

(d) at least one container for packaging the nucleotide incorporating biocatalyst, the labeled 2′

-terminator nucleotide, and the set of instructions.

111. The kit of claim 102, wherein the nucleotide incorporating biocatalyst comprises an enzyme selected from the group consisting of:

a polymerase, a terminal transferase, a reverse transcriptase, a polynucleotide phosphorylase, and a telomerase.

112. The kit of claim 111, wherein the enzyme comprises a mutation that enhances incorporation of ribonucleotides.

113. The kit of claim 111, wherein the enzyme comprises a mutation that reduces or eliminates 5′

-3′

exonuclease activity.

114. The kit of claim 111, wherein the enzyme comprises a mutation that enhances incorporation of 2′

-modified analogs of ribonucleotides.

115. The kit of claim 111, wherein the enzyme comprises a 3′

-5′

exonuclease activity.

116. The kit of claim 111, wherein the enzyme comprises a thermostable enzyme.

117. The kit of claim 111, wherein the enzyme is derived from an organism that is selected from the group consisting of:

Thermus antranikianii, Thermus aquaticus, Thermus caldophilus, Thermus chliarophilus, Thermus filiformis, Thermus flavus, Thermus igniterrae, Thermus lacteus, Thermus oshimai, Thermus ruber, Thermus rubens, Thermus scotoductus, Thermus silvanus, Thermus species Z05, Thermus species sps 17, Thermus thermophilus, Thermotoga maritima, Thermotoga neapolitana, Thermosipho africanus, Anaerocellum thermophilum, Bacillus caldotenax, and Bacillus stearothermophilus.

118. The kit of claim 111, wherein the enzyme is modified.

119. The kit of claim 118, wherein the modified enzyme comprises one or more of:

is a G46E E678G CS5 DNA polymerase, a G46E E678G CS6 DNA polymerase, an E615G Taq DNA polymerase, a Δ

ZO5R polymerase, or a G46E L329A E678G CS5 DNA polymerase.

120. The kit of claim 102, wherein the label comprises a radioisotope, a fluorescent dye, or a mass-modifying group.

121. The kit of claim 102, wherein the nucleic acid comprises a primer nucleic acid and the kit further comprises a template nucleic acid and the primer nucleic acid, which primer nucleic acid is complementary to at least a subsequence of the template nucleic acid.

122. The kit of claim 121, wherein the template nucleic acid or the primer nucleic acid is attached to a solid support.

123. The kit of claim 121, wherein the primer nucleic acid comprises a label.

124. The kit of claim 123, wherein the label comprises a radioisotope, a fluorescent dye, or a mass-modifying group.

125. A system for extending a nucleic acid, comprising:

(a) at least one container comprising a labeled 2′

-terminator nucleotide; and

at least one of

(b) at least one thermal modulator operably connected to the container to modulate temperature in the container; and

,

(c) at least one fluid transfer component that transfers fluid to and/or from the container.

126. The system of claim 125, wherein the system comprises a plurality of containers.

127. The system of claim 125, further comprising at least one detector operably connected to the container to detect detectable signals produced in the container.

128. The system of claim 125, further comprising at least one controller operably connected to the thermal modulator to effect modulation of the temperature in the container and/or to the fluid transfer component to effect transfer of the fluid to and/or from the container.