Nucleotide transient binding for sequencing methods

• US 8,632,975 B2
• Filed: 05/28/2010
• Issued: 01/21/2014
• Est. Priority Date: 06/05/2009
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Chen, Cheng-Yao, Luo, Guobin, Previte, Michael, Temirov, Jamshid, Nikiforov, Theo, Zhou, Zhaohui, Sun, Hongye, Nishimura, Stefanie Yukiko, Wang, Hongyi, Peris, Marian, Rosenblum, Barnett B., Phelan, Michael, Vander Horn, Peter B., Wang, Yufang
• Primary Examiner(s): Babic, Christopher M
• Assistant Examiner(s): PRIEST, AARON A
• Application Number:
  US12/790,760
• Publication Number:
  US 20100330570A1
• Time in Patent Office: 1,334 Days
• Field of Search: None
• US Class Current: 435/6.12
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6872 involving mass spectrometry
• CPC Class Codes: C12Q 2537/157 A reaction step characterised by the number of molecules incorporated or released
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2563/137 Metal/ion, e.g. metal label
• CPC Class Codes: C12Q 2565/30 Detection characterised by liberation or release of label
• CPC Class Codes: C12Q 2565/632 being a surface enhanced, e.g. resonance, Raman spectrometer
• CPC Class Codes: C12Y 207/07007 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: G01N 2500/00 Screening for compounds of potential therapeutic value

Abstract

Provided herein are compositions and systems for use in polymerase-dependent, nucleotide transient-binding methods. The methods are useful for deducing the sequence of a template nucleic acid molecule and single nucleotide polymorphism (SNP) analyses. The methods rely on the fact that the polymerase transient-binding time for a complementary nucleotide is longer compared to that of a non-complementary nucleotide. The labeled nucleotides transiently-binds the polymerase in a template-dependent manner, but does not incorporate. The methods are conducted under any reaction condition that permits transient binding of a complementary or non-complementary nucleotide to a polymerase, and inhibits nucleotide incorporation.

Claims

1. A method for identifying a nucleotide bound to a polymerase, comprising:

a) transiently binding, without polymerizing, a first type of nucleotide to a first polymerase in the presence of a cation that inhibits nucleotide incorporation by the polymerase, wherein the first polymerase is bound to a template nucleic acid molecule that is bound to a polymerization initiation site, wherein the first polymerase lacks exonuclease activity, and wherein the first type of nucleotide includes a detectable moiety;

b) detecting the first type of transiently-bound nucleotide; and

c) identifying the first type of transiently-bound nucleotide.

2. The method of claim 1 further comprising:

a) removing the transiently-bound first type of nucleotide; and

b) contacting the complex with a second type of nucleotide under suitable conditions for the first polymerase to polymerize the second type of nucleotide in a template-dependent manner.

3. The method of claim 2, wherein the suitable conditions in step (b) of claim 2 further include any one or more of the following:

(i) including a metal cation that permits nucleotide incorporation and/or reducing the levels or omission of a cation that inhibits nucleotide incorporation;

(ii) the first polymerase selectively binds the second type of nucleotide in a template-dependent manner and polymerizes the bound second type of nucleotide;

(iii) the second type of nucleotide is an incorporatable nucleotide; and

(iv) the polymerization initiation is an extendible polymerization initiation site.

4. The method of claim 1, wherein the template nucleic acid molecule is linked to a surface.

5. The method of claim 1, wherein the first type of nucleotide includes an optically detectable label, and wherein the method further includes exciting the optically detectable label with electromagnetic radiation.

6. The method of claim 1, wherein the cation that inhibits nucleotide incorporation is calcium, scandium, titanium, vanadium, chromium, iron, cobalt, nickel, copper, zinc, gallium, germanium, arsenic, selenium, rhodium, or strontium.

7. The method of claim 1, wherein the first polymerase is a DNA-dependent polymerase, RNA-dependent polymerase, or a reverse transcriptase.

8. The method of claim 1, wherein the first polymerase is an RB69 polymerase, phi29 polymerase, B103 polymerase, or Klenow fragment polymerase.

9. The method of claim 1, wherein the first polymerase is an RB69 polymerase according to SEQ ID NO:

5.

10. The method of claim 1, wherein the second polymerase is the same type or a different type as the first polymerase.

11. The method of claim 1, wherein the first type of nucleotide in step (a) comprises 3-10 phosphate groups.

12. The method of claim 1, wherein the first type of nucleotide in step (a) includes an adenosine, guanosine, cytidine, thymidine, or uridine moiety.

13. The method of claim 1, wherein the first type of nucleotide comprises a fluorescent label linked to the base of the nucleotide or linked to the terminal phosphate group of the nucleotide.

14. The method of claim 13, wherein the label comprises a fluorescent dye.

15. The method of claim 13, wherein the label comprises an energy transfer acceptor reporter moiety.

16. The method of claim 1, further including contacting the complex with at least two different types of nucleotides in step (a).

17. The method of claim 16, wherein each different type of nucleotide includes a different type of label.

18. The method of claim 1, wherein the first polymerase comprises an energy transfer donor reporter moiety.

19. The method of claim 18, wherein the energy transfer donor reporter moiety is an inorganic nanoparticle or a fluorophore.

20. The method of claim 1, wherein the first polymerase comprises a label including an energy transfer donor reporter moiety and the first type of transiently-bound nucleotide comprises an energy transfer acceptor reporter moiety.

21. The method of claim 20, wherein the transiently-bound labeled nucleotide produces a FRET signal.

22. The method of claim 21, wherein the signal from the transiently-bound labeled nucleotide is optically detectable.

23. The method of claim 1, wherein the first type of nucleotide includes an extrinsic label containing a fluorescent moiety.

24. The method of claim 23, wherein the first type of nucleotide includes an extrinsic label containing an energy transfer acceptor reporter moiety.

25. The method of claim 18, wherein the energy transfer donor reporter moiety is an inorganic nanoparticle or a fluorophore.

26. The method of claim 1, wherein the first polymerase comprises an energy transfer donor reporter moiety and the first type of nucleotide comprises an energy transfer acceptor reporter moiety.

27. The method of claim 22, wherein the signal emitted by the transiently-bound labeled nucleotide is a FRET signal.

28. The method of claim 1, wherein the template nucleic acid molecule is a DNA, RNA, or DNA/RNA hybrid molecule.

82. The method of claim 1, wherein the transiently binding is performed in a reaction mixture that does not include magnesium.

29. A method for identifying a nucleotide bound to a polymerase, comprising:

transiently binding, without polymerizing, at least a first type of a nucleotide including a detectable moiety to an immobilized complex including a first polymerase, a template nucleic acid strand, and a polymerization initiation site, wherein the transiently binding includes forming a reaction mixture including the first type of nucleotide and the first polymerase and having a concentration of cation that inhibits nucleotide polymerization by the polymerase;

b. identifying the first type of nucleotide transiently-bound to the first polymerase;

c. removing the transiently-bound first type of nucleotide;

d. contacting the complex with at least a second type of nucleotide under suitable conditions for the first polymerase to polymerize the second type of nucleotide at the polymerization initiation site in a template-dependent manner; and

e. repeating steps (a)-(c) at least once.

30. The method of claim 29, wherein the forming in step (a) of claim 29 includes any one or more of the following:

(i) reducing the levels or omission of a metal cation that permits nucleotide incorporation and/or addition of a cation that inhibits nucleotide incorporation.

31. The method of claim 29, wherein the suitable conditions in step (d) comprise any one or more of the following:

(i) including a metal cation that permits nucleotide incorporation and/or reducing the levels or omission of a cation that inhibits nucleotide incorporation;

(ii) using a first polymerase which selectively binds the second type of nucleotide in a template-dependent manner and polymerizes the bound second type of nucleotide;

(iii) using a second type of nucleotide which includes an incorporatable nucleotide; and

/or (iv) using a polymerization initiation site having an extendible polymerization initiation site.

83. The method of claim 29, wherein the reaction mixture does not include magnesium.

32. A method for obtaining sequence information from a template nucleic acid molecule, comprising:

(a) forming a reaction mixture including a polymerase and a template nucleic acid molecule, wherein the reaction mixture includes a concentration of cation at which nucleotide polymerization by the polymerase is inhibited;

(b) contacting, within the reaction mixture, a first type of nucleotide containing a detectable moiety with the polymerase and transiently binding the first type of nucleotide to the polymerase in a template-dependent manner without polymerizing the transiently bound nucleotide at the polymerization initiation site by the polymerase,

(c) detecting a signal generated by the detectable moiety while the first type of nucleotide is transiently bound to the polymerase;

(d) removing the transiently bound nucleotide from the polymerase; and

(e) contacting the polymerase with a second type of nucleotide and polymerizing the second type of nucleotide at the polymerization initiation site using the polymerase.

33. The method of claim 32, further including performing the detecting step (b) without removing unbound nucleotide from the reaction mixture.

34. The method of claim 32, wherein the first type and second type of nucleotide are different types.

35. The method of claim 32, wherein the first type and second type of nucleotide are the same type.

36. The method of claim 32, wherein the template includes no more than a single nucleic acid molecule.

37. The method of claim 32, further including:

obtaining sequence information from a plurality of substantially identical template nucleic acid molecules by simultaneously subjecting them to the method of claim 32 within the same reaction mixture.

38. The method of claim 32, further including:

obtaining sequence information from a plurality of different template nucleic acid molecules by simultaneously subjecting them to the method of claim 32 in parallel, optionally within the same reaction mixture.

39. The method of claim 32, wherein the first type of nucleotide includes an extrinsic label.

40. The method of claim 39, wherein the first type of nucleotide includes a fluorescent label.

41. The method of claim 39, wherein the first type of nucleotide includes a FRET acceptor.

42. The method of claim 32, wherein the first type of nucleotide includes an extrinsic fluorescent label.

43. The method of claim 32, wherein the second type of nucleotide includes an extrinsic fluorescent label.

44. The method of claim 32, wherein the first type of nucleotide does not include an extrinsic fluorescent label.

45. The method of claim 32, wherein the second type of nucleotide does not include an extrinsic fluorescent label.

46. The method of claim 32, wherein the first or the second type of nucleotide is not optically detectable.

47. The method of claim 32, wherein the first type of nucleotide includes an extrinsic fluorescent label and the second type does not.

48. The method of claim 32, wherein the transiently binding in step (b) includes contacting the polymerase with a sufficient amount of a divalent cation to inhibit nucleotide incorporation by the first polymerase.

49. The method of claim 48, wherein the divalent cation includes calcium or magnesium.

50. The method of claim 32, further including repeating steps (a), (b) and (c) at least once.

51. The method of claim 50, further including repeating steps (d) and (e) at least once.

52. The method of claim 32, wherein the transiently binding without polymerizing includes not forming any covalent bond between the transiently bound nucleotide and a free 3′

-OH end of a primer or nucleic acid molecule.

76. The method of claim 32, wherein the transiently binding in step (b) of claim 32 includes forming a reaction mixture that includes reduced concentrations of a cation required for nucleotide polymerization by the polymerase, and wherein the concentration of the cation is reduced sufficiently to inhibit nucleotide polymerization by the polymerase.

77. The method of claim 76, wherein the transiently binding in step (b) of claim 32 includes forming a reaction mixture that includes a cation that inhibits nucleotide polymerization by the polymerase, and wherein the cation is present in an amount sufficient to inhibit nucleotide polymerization.

84. The method of claim 32, wherein the reaction mixture does not include magnesium.

53. A method for obtaining sequence information from a template nucleic acid molecule, comprising:

(a) transiently binding, without polymerizing, a first type of nucleotide having a detectable moiety to a polymerase, wherein the transiently binding includes forming a reaction mixture having a concentration of cation at which nucleotide polymerization by the polymerase is inhibited; and

(b) detecting a signal, the signal being generated by the detectable moiety during the transiently binding; and

(c) identifying the first type of nucleotide.

54. The method of claim 53, further including performing the detecting step (b) without removing unbound nucleotide from the reaction mixture.

55. The method of claim 53, further including removing the transiently bound nucleotide from the polymerase.

56. The method of claim 54, further including transiently binding a second type of nucleotide using the polymerase.

57. The method of claim 54, further including polymerizing a second type of nucleotide using the polymerase.

58. The method of claim 57, wherein the first type and second type of nucleotide are different types.

59. The method of claim 57, wherein the first type and second type of nucleotide are the same type.

60. The method of claim 57, wherein the polymerase is bound to a template nucleic acid molecule.

61. The method of claim 60, wherein the template includes no more than a single nucleic acid molecule.

62. The method of claim 53, further including:

obtaining sequence information from a plurality of substantially identical template nucleic acid molecules by simultaneously subjecting them to the method of claim 53 within the same reaction mixture.

63. The method of claim 53, further including:

obtaining sequence information from a plurality of different template nucleic acid molecules by simultaneously subjecting them to the method of claim 53 in parallel, optionally within the same reaction mixture.

64. The method of claim 53, wherein the first type of nucleotide includes an extrinsic label.

65. The method of claim 64, wherein the first type of nucleotide includes a fluorescent label.

66. The method of claim 65, wherein the first type of nucleotide includes a FRET acceptor.

67. The method of claim 57, wherein the second type of nucleotide includes an extrinsic fluorescent label.

68. The method of claim 53, wherein the first type of nucleotide does not include an extrinsic fluorescent label.

69. The method of claim 57, wherein the second type of nucleotide does not include an extrinsic fluorescent label.

70. The method of claim 57, wherein the first or the second type of nucleotide is not optically detectable.

71. The method of claim 57, wherein the first type of nucleotide includes an extrinsic fluorescent label and the second type does not.

72. The method of claim 53, wherein the transiently binding in step (b) includes contacting the polymerase with a sufficient amount of a cation to inhibit nucleotide incorporation by the first polymerase.

73. The method of claim 72, wherein the divalent cation includes calcium.

74. The method of claim 53, further including repeating steps (a), (b) and (c) at least once.

75. The method of claim 56, further including repeating the removing and the polymerizing the second nucleotide at least once.

78. The method of claim 53, wherein the transiently binding in step (b) of claim 53 includes forming a reaction mixture that includes reduced concentrations of a cation required for nucleotide polymerization by the polymerase, and wherein the concentration of the cation is reduced sufficiently to inhibit nucleotide polymerization by the polymerase.

79. The method of claim 78, wherein the cation includes magnesium.

80. The method of claim 53, wherein the transiently binding in step (b) of claim 53 includes forming a reaction mixture that includes a cation that inhibits nucleotide polymerization by the polymerase, and wherein the cation is present in an amount sufficient to inhibit nucleotide polymerization.

81. The method of claim 78, wherein the cation includes calcium.

85. The method of claim 53, wherein the reaction mixture does not include magnesium.