Method for drying dye-terminator sequencing reagents

• US 20040072167A1
• Filed: 10/15/2002
• Published: 04/15/2004
• Est. Priority Date: 10/15/2002
• Status: Active Grant

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Inventors: Ma, Peter, Spurgeon, Sandra L., Ruhfel, Robert E., Perry, Kathleen M.
• Granted Patent: US 7,407,747 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C12Q 1/6869 Methods for sequencing

Abstract

A dry composition comprising a nucleotide linked to a fluorescent dye is provided. A method of making a dry composition comprising the step of drying a solution comprising a nucleotide linked to a fluorescent dye is provided. A method is provided for performing a reaction on a nucleic acid substrate comprising: (a) providing a vessel containing a dried composition comprising a nucleotide linked to a fluorescent dye, and (b) adding to the vessel an aqueous solution and the nucleic acid substrate. A kit is provided for determining the sequence of a nucleic acid, the kit comprising a dry composition comprising a nucleotide linked to a fluorescent dye.

Claims

1. A dry composition comprising a nucleotide linked to a fluorescent dye.

2. The composition of claim 1 wherein the nucleotide is part of a polynucleotide.

3. The composition of claim 1 wherein the nucleotide is a mononucleotide.

4. The composition of claim 1 wherein the nucleotide is a chain terminator nucleotide.

5. The composition of claim 4 further comprising three other chain terminator nucleotides, each of the four chain terminator nucleotides being linked to a fluorescent dye, wherein each of the four chain terminator nucleotides is linked to a different fluorescent dye.

6. The composition of claim 5 wherein the chain terminator nucleotides are 2′

,3′

-dideoxynucleotides.

7. The composition of claim 5 further comprising four nucleoside triphosphates.

8. The composition of claim 1 further comprising a thermostable nucleic acid polymerase.

9. The composition of claim 8 wherein the polymerase is Taq DNA polymerase.

10. The composition of claim 9 wherein the composition further comprises another nucleic acid polymerase.

11. The composition of claim 1 wherein the composition is substantially free of glycerol.

12. The composition of claim 1 or 8 further comprising a cryoprotectant.

13. The composition of claim 12 wherein the cryoprotectant is trehalose.

14. The composition of claim 13 wherein the cryoprotectant is sucrose.

15. The composition of claim 1 wherein the dye is an energy transfer dye comprising a donor connected to an acceptor by a linker.

16. The composition of claim 15 wherein the linker comprises an alkene, diene, alkyne, unsaturated five-membered ring, unsaturated six-membered ring, or a fused ring system.

17. The composition of claim 16 wherein the dye has the structure

18. The composition of claim 15 wherein the donor is a fluorescein, rhodamine, or asymmetric benzoxanthene dye.

19. The composition of claim 18 wherein the donor is a carboxyfluorescein;

4,7-dichlorofluorescein;

asymmetric benzoxanthene;

rhodamine;

4,7-dichlororhodamine;

carboxyrhodamine;

carboxy R110;

carboxy R6G;

or carboxy-X-rhodamine dye;

or is Cy5.

20. The composition of claim 15 wherein the acceptor is a xanthene, cyanine, phthalocyanine, or squaraine dye.

21. The composition of claim 20 wherein the acceptor is a 4,7-dichlorofluorescein;

asymmetric benzoxanthene;

rhodamine;

4,7-dichlororhodamine;

carboxyrhodamine;

carboxy R110;

carboxy R6G;

or carboxy-X-rhodamine dye;

or is Cy5.

22. The composition of claim 1 wherein the composition comprises less than 20% solvent content by weight.

23. The composition of claim 1 wherein the composition comprises less than 20% water content by weight.

24. The composition of claim 1 wherein the composition comprises less than 5% solvent content by weight.

25. The composition of claim 1 wherein the composition comprises less than 5% water content by weight.

26. The composition of claim 1 wherein the composition comprises less than 2% solvent content by weight.

27. The composition of claim 1 wherein the composition comprises less than 2% water content by weight.

28. A method of making the composition of claim 1 comprising the step of drying a solution comprising the nucleotide linked to a fluorescent dye.

29. The method of claim 28 wherein the nucleotide is part of a polynucleotide.

30. The method of claim 28 wherein the nucleotide is a mononucleotide.

31. The method of claim 28 wherein the nucleotide is a chain terminator nucleotide.

32. The method of claim 31 further comprising drying one or more solutions collectively comprising three other chain terminator nucleotides, each of the four chain terminator nucleotides being linked to a fluorescent dye, wherein each of the four chain terminator nucleotides is linked to a different fluorescent dye.

33. The composition of claim 32 wherein the chain terminator nucleotides are 2′

,3′

-dideoxynucleotides.

34. The method of claim 32 further comprising drying one or more solutions collectively comprising four nucleoside triphosphates.

35. The method of claim 28 further comprising drying a solution comprising a thermostable nucleic acid polymerase.

36. The method of claim 28 or 35 wherein the solutions are aqueous.

37. The method of claim 35 wherein the polymerase is Taq DNA polymerase.

38. The method of claim 35 wherein the solution comprising a thermostable nucleic acid polymerse is substantially free of glycerol.

39. The method of claim 35 wherein the solution comprising a thermostable nucleic acid polymerase further comprises a cryoprotectant.

40. The method of claim 39 wherein the cryoprotectant is trehalose.

41. The method of claim 39 wherein the cryoprotectant is sucrose.

42. The method of claim 28 wherein the temperature of the solution during at least a portion of the drying step is less than 0°

C.

43. The method of claim 35 wherein the temperature of the solution comprising the thermostable nucleic acid polymerase during at least a portion of the step of drying the solution comprising the thermostable polymerase is less than 0°

C.

44. The method of claim 28 wherein the dye is an energy transfer dye comprising a donor connected to an acceptor by a linker.

45. The method of claim 44 wherein the linker comprises an alkene, diene, alkyne, unsaturated five-membered ring, unsaturated six-membered ring, or a fused ring system.

46. The method of claim 45 wherein the dye has the structure

47. The method of claim 44 wherein the donor is a fluorescein, rhodamine, or asymmetric benzoxanthene dye.

48. The method of claim 47 wherein the donor is a carboxyfluorescein;

4,7-dichlorofluorescein;

asymmetric benzoxanthene;

rhodamine;

4,7-dichlororhodamine;

carboxyrhodamine;

carboxy R110;

carboxy R6G;

or carboxy-X-rhodamine dye;

or is Cy5.

49. The method of claim 44 wherein the acceptor is a xanthene, cyanine, phthalocyanine, or squaraine dye.

50. The method of claim 49 wherein the acceptor is a 4,7-dichlorofluorescein;

asymmetric benzoxanthene;

rhodamine;

4,7-dichlororhodamine;

carboxyrhodamine;

carboxy R110;

carboxy R6G;

or carboxy-X-rhodamine dye;

or is Cy5.

51. The method of claim 28 wherein the dried composition comprises less than 20% solvent content by weight.

52. A method of performing a reaction on a nucleic acid substrate comprising:

(a) providing a vessel containing a dry composition comprising a nucleotide linked to a fluorescent dye, and

(b) adding to the vessel an aqueous solution and the nucleic acid substrate.

53. The method of claim 52 wherein the reaction is a nucleic acid sequencing reaction, and wherein the method further comprises extending a primer hybridized to the nucleic acid substrate with a nucleic acid polymerase and determining the nucleotide sequence of at least a portion of the nucleic acid substrate.

54. The method of claim 52 wherein the nucleotide is part of a polynucleotide.

55. The method of claim 52 wherein the nucleotide is a mononucleotide.

56. The method of claim 52 wherein the nucleotide is a chain terminator nucleotide.

57. The method of claim 56 wherein the dry composition further comprises three other chain terminator nucleotides, each of the four chain terminator nucleotides being linked to a fluorescent dye, wherein each of the four chain terminator nucleotides is linked to a different fluorescent dye.

58. The method of claim 57 wherein the chain terminator nucleotides are 2′

,3′

-dideoxynucleotides.

59. The method of claim 57 wherein the dry composition further comprises four nucleoside triphosphates.

60. The method of claim 52 wherein the dry composition further comprises a thermostable nucleic acid polymerase.

61. The method of claim 60 wherein the polymerase is Taq DNA polymerase.

62. The method of claim 52 wherein the dry composition is substantially free of glycerol.

63. The method of claim 52 wherein the dry composition further comprises a cryoprotectant.

64. The method of claim 63 wherein the cryoprotectant is trehalose.

65. The method of claim 63 wherein the cryoprotectant is sucrose.

66. The method of claim 52 wherein the dye is an energy transfer dye comprising a donor connected to an acceptor by a linker.

67. The method of claim 66 wherein the linker comprises an alkene, diene, alkyne, unsaturated five-membered ring, unsaturated six-membered ring, or a fused ring system.

68. The method of claim 66 wherein the dye has the structure

69. The method of claim 66 wherein the donor is a fluorescein, rhodamine, or asymmetric benzoxanthene dye.

70. The method of claim 69 wherein the donor is a carboxyfluorescein;

4,7-dichlorofluorescein;

asymmetric benzoxanthene;

rhodamine;

4,7-dichlororhodamine;

carboxyrhodamine;

carboxy R110;

carboxy R6G;

or carboxy-X-rhodamine dye;

or is Cy5.

71. The method of claim 66 wherein the acceptor is a xanthene, cyanine, phthalocyanine, or squaraine dye.

72. The method of claim 71 wherein the acceptor is a 4,7-dichlorofluorescein;

asymmetric benzoxanthene;

rhodamine;

4,7-dichlororhodamine;

carboxyrhodamine;

carboxy R110;

carboxy R6G;

or carboxy-X-rhodamine dye;

or is Cy5.

73. The method of claim 52 wherein the nucleic acid substrate is unconcentrated nucleic acid derived from lysed whole cells.

74. The method of claim 73 wherein the cells are bacterial cells.

75. The method of claim 73 wherein the cells are animal, fungal, or plant cells.

76. The method of claim 52 wherein the nucleic acid substrate is derived from lysed whole cells and is concentrated by a factor of no more than 3 relative to its concentration in an extracellular extract immediately after lysis of the cells.

77. The method of claim 52 wherein the dry composition comprises less than 20% solvent content by weight.

78. A kit for determining the sequence of a nucleic acid, the kit comprising a dry composition comprising a nucleotide linked to a fluorescent dye.

79. The kit of claim 78 wherein the nucleotide is part of a polynucleotide.

80. The kit of claim 78 wherein the nucleotide is a mononucleotide.

81. The kit of claim 78 wherein the nucleotide is a chain terminator nucleotide.

82. The kit of claim 81 wherein the dry composition further comprises three other chain terminator nucleotides, each of the four chain terminator nucleotides being linked to a fluorescent dye, wherein each of the four chain terminator nucleotides is linked to a different fluorescent dye.

83. The kit of claim 82 wherein the chain terminator nucleotides are 2′

,3′

-dideoxynucleotides.

84. The kit of claim 82 wherein the dry composition further comprises four nucleoside triphosphates.

85. The kit of claim 78 wherein the dry composition further comprises a thermostable nucleic acid polymerase.

86. The kit of claim 85 wherein the polymerase is Taq DNA polymerase.

87. The kit of claim 78 wherein the dry composition is substantially free of glycerol.

88. The kit of claim 78 wherein the dry composition further comprises a cryoprotectant.

89. The kit of claim 88 wherein the cryoprotectant is trehalose.

90. The kit of claim 88 wherein the cryoprotectant is sucrose.

91. The kit of claim 78 wherein the dye is an energy transfer dye comprising a donor connected to an acceptor by a linker.

92. The kit of claim 91 wherein the linker comprises an alkene, diene, alkyne, unsaturated five-membered ring, unsaturated six-membered ring, or a fused ring system.

93. The kit of claim 92 wherein the dye has the structure

94. The kit of claim 91 wherein the donor is a fluorescein, rhodamine, or asymmetric benzoxanthene dye.

95. The kit of claim 94 wherein the donor is a carboxyfluorescein;

4,7-dichlorofluorescein;

asymmetric benzoxanthene;

rhodamine;

4,7-dichlororhodamine;

carboxyrhodamine;

carboxy R110;

carboxy R6G;

or carboxy-X-rhodamine dye;

or is Cy5.

96. The kit of claim 91 wherein the acceptor is a xanthene, cyanine, phthalocyanine, or squaraine dye.

97. The kit of claim 96 wherein the acceptor is a 4,7-dichlorofluorescein;

asymmetric benzoxanthene;

rhodamine;

4,7-dichlororhodamine;

carboxyrhodamine;

carboxy R110;

carboxy R6G;

or carboxy-X-rhodamine dye;

or is Cy5.

98. The kit of claim 78 wherein the kit comprises three or more substantially identical dry compositions comprising the nucleotide, the dry compositions arranged in a regular pattern on a solid substrate.

99. The kit of claim 78 wherein the dry composition comprises less than 20% solvent content by weight.