Compositions and methods for sequence determination

• US 20100255463A1
• Filed: 12/29/2006
• Published: 10/07/2010
• Est. Priority Date: 07/07/2000
• Status: Abandoned Application

• Current Assignee: James Briggs, Richard Willson, Shiao-Chun Tu, Susan Harsin, Xiaolian Gao
• Original Assignee: James Briggs, Richard Willson, Shiao-Chun Tu, Susan Harsin, Xiaolian Gao
• Inventors: Harsin, Susan, Willson, Richard, Briggs, James, Tu, Shiao-Chun, Gao, Xiaolian
• Application Number:
  US11/648,184
• Publication Number:
  US 20100255463A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels
• CPC Class Codes: C12Q 2565/133 conformational analysis
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)

Abstract

A sequencing methodology is disclosed that allows a single DNA or RNA molecule or portion thereof to be sequenced directly and in substantially real time. The methodology involves engineering a polymerase and/or dNTPs with atomic and/or molecular tags that have a detectable property that is monitored by a detection system.

Claims

1. (canceled)

2. (canceled)

3. (canceled)

4. (canceled)

5. (canceled)

6. (canceled)

7. (canceled)

8. (canceled)

9. (canceled)

10. A composition comprising a polymerizing agent including a fluorescent tag, a nucleic acid template, a primer adapted to hybridize to a complementary portion of the template to form a primer-template duplex and monomers for the polymerizing agent, where one monomer type includes a first quencher having a first quenching efficiency, where the fluorescent tag is quenched before, during and/or after the incorporation of each first quencher modified monomer in a sequence of monomer incorporations due to an interaction between the fluorescent tag and the first quencher as each first quencher modified monomer is incorporated.

14. The composition of claim 10, wherein the polymerizing agent is a polymerase or reverse transcriptase.

17. The composition of claim 10, wherein the monomers comprise dNTPs and the first quencher is covalently bonded to a p, γ

or terminal phosphate group of one monomer type.

19. The composition of claim 10, wherein the fluorescent tag has a higher or unquenched fluorescent intensity prior to a monomer incorporation cycle and a lower or quenched fluorescent intensity during each incorporation of a first quencher labeled monomer in the sequence of monomer incorporations due to the quenching efficiency of the first quencher.

35. The composition of claim 10, further comprising a second monomer type including a second quencher having a second quenching efficiency, where the first and second quenchers are the same or different and the first and second quenching efficiencies are the same or different and where the fluorescent tag is quenched during each incorporation of a second modified monomer type in a sequence of monomer incorporations due to an interaction between the fluorescent tag and the second quencher first or.

36. The composition of claim 10, further comprising a second monomer type include a second quencher having a second quenching efficiency and a third monomer type including a third quencher having a third quenching efficiency, where the first, second and third quenchers are the same or different and the first, second and third quenching efficiencies are the same or different and where the fluorescent tag is quenched during each incorporation of a second or third monomer type in a sequence of monomer incorporations due to an interaction between the fluorescent tag and the second or third.

37. The composition of claim 10, further comprising a second monomer type including a second quencher having a second quenching efficiency, a third monomer type including a third quencher having a third quenching efficiency, and a fourth monomer type including a fourth quencher having a fourth quenching efficiency, where the first, second, third and fourth quenchers are the same or different and the first, second, third and fourth quenching efficiencies are the same or different and where the fluorescent tag is quenched during each monomer incorporation of a second, third or fourth monomer type in a sequence of monomer incorporations due to an interaction between the fluorescent tag and the second, third or fourth quencher.

11. (canceled)

12. (canceled)

13. (canceled)

15. The composition of claim 13, wherein the polymerase is selected from the group consisting of Tag DNA polymerase I, T7 DNA polymerase, Sequenase, and the Klenow fragment from E. coli DNA polymerase I and wherein the reverse transcriptase comprises HIV-1 reverse transcriptase.

16. (canceled)

18. (canceled)

20. (canceled)

21. (canceled)

22. (canceled)

23. (canceled)

24. (canceled)

25. (canceled)

26. (canceled)

27. (canceled)

28. (canceled)

29. (canceled)

30. (canceled)

31. (canceled)

32. (canceled)

33. (canceled)

34. (canceled)

38. A composition:

comprising a polymerizing agent including a fluorescent tag,

a nucleic acid template, a primer adapted to hybridize to a complementary portion of the template to form a primer-template duplex, and

monomers for the polymerizing agent, where one monomer type includes a first quencher covalently bonded to a p, γ

or terminal phosphate group of one monomer type and where the first quencher has a first quenching efficiency,

where the fluorescent tag is quenched before, during and/or after each monomer incorporation of a first quencher modified monomer in a sequence of monomer incorporations due to an interaction between the fluorescent tag and the first quencher as each first quencher modified monomer is incorporated.

39. The composition of claim 38, further comprising a second monomer type including a second quencher having a second quenching efficiency, where the first and second quenchers are the same or different and the first and second quenching efficiencies are the same or different and where the fluorescent tag is quenched before, during and/or after each incorporation of a second monomer type in a sequence of monomer incorporations due to an interaction between the fluorescent tag and the second quencher.

40. The composition of claim 38, further comprising a second monomer type including a second quencher having a second quenching efficiency and a third monomer type including a third quencher having a third quenching efficiency, where the first, second and third quenchers are the same or different and the first, second and third quenching efficiencies are the same or different and where the fluorescent tag is quenched during each incorporation of a second or third monomer type in a sequence of monomer incorporations due to an interaction between the fluorescent tag and the second or third quencher.

41. The composition of claim 38, further comprising a second monomer type including a second quencher having a second quenching efficiency, a third monomer type including a third quencher having a third quenching efficiency, and a fourth monomer type including a fourth quencher having a fourth quenching efficiency, where the first, second, third and fourth quenchers are the same or different and the first, second, third and fourth quenching efficiencies are the same or different and where the fluorescent tag is quenched during each incorporation of a second, third or fourth quencher modified monomer in a sequence of monomer incorporations due to an interaction between the fluorescent tag and the second, third or fourth quencher.

42. The composition of claim 38, wherein the polymerizing agent is a polymerase or reverse transcriptase.

43. The composition of claim 42, wherein the polymerase is selected from the group consisting of Tag DNA polymerase I, T7 DNA polymerase, Sequenase, and the Klenow fragment from E. coli DNA polymerase I and wherein the reverse transcriptase comprises HIV-1 reverse transcriptase.

44. A composition:

comprising a polymerizing agent including a fluorescent tag,

a nucleic acid template, a primer adapted to hybridize to a complementary portion of the template to form a primer-template duplex, and

monomers for the polymerizing agent, where one monomer type includes a first quencher covalently bonded to a β

phosphate group of one monomer type and where the first quencher has a first quenching efficiency,

where the fluorescent tag is quenched during each monomer incorporation of a first quencher modified monomer in a sequence of monomer incorporations due to an interaction between the fluorescent tag and the first quencher as each first quencher modified monomer is incorporated.

45. The composition of claim 44, further comprising a second monomer type including a second quencher having a second quenching efficiency, where the first and second quenchers are the same or different and the first and second quenching efficiencies are the same or different and where the fluorescent tag is quenched during each incorporation of a second monomer type in a sequence of monomer incorporations due to an interaction between the fluorescent tag and the second quencher.

46. The composition of claim 44, further comprising a second monomer type include a second quencher having a second quenching efficiency and a third monomer type include a second quencher having a third quenching efficiency, where the first, second and third quenchers are the same or different and the first, second and third quenching efficiencies are the same or different and where the fluorescent tag is quenched during each monomer incorporation of a first, second or third quencher modified monomer in a sequence of monomer incorporations due to an interaction between the fluorescent tag and the second or third quencher.

47. The composition of claim 44, further comprising a second monomer type including a second quencher having a second quenching efficiency, a third monomer type including a third quencher having a third quenching efficiency, and a fourth monomer type including a fourth quencher having a fourth quenching efficiency, where the first, second, third and fourth quenchers are the same or different and the first, second, third and fourth quenching efficiencies are the same or different and where the fluorescent tag is quenched during each incorporation of a second, third or fourth monomer type in a sequence of monomer incorporations due to an interaction between the fluorescent tag and the second, third or fourth quencher.

48. The composition of claim 44, wherein the polymerizing agent is a polymerase or reverse transcriptase.

49. The composition of claim 48, wherein the polymerase is selected from the group consisting of Tag DNA polymerase I, T7 DNA polymerase, Sequenase, and the Klenow fragment from E. coli DNA polymerase I and wherein the reverse transcriptase comprises HIV-1 reverse transcriptase.

50. A composition:

comprising a polymerizing agent including a fluorescent tag,

a nucleic acid template, a primer adapted to hybridize to a complementary portion of the template to form a primer-template duplex, and

monomers for the polymerizing agent, where one monomer type includes a first quencher covalently bonded to a γ

phosphate group of one monomer type and where the first quencher has a first quenching efficiency,

where the fluorescent tag is quenched during each monomer incorporation of a first quencher modified monomer in a sequence of monomer incorporations due to an interaction between the fluorescent tag and the first quencher as each first quencher modified monomer is incorporated.

51. The composition of claim 50, further comprising a second monomer type including a second quencher having a second quenching efficiency, where the first and second quenchers are the same or different and the first and second quenching efficiencies are the same or different and where the fluorescent tag is quenched during each incorporation of a second monomer type in a sequence of monomer incorporations due to an interaction between the fluorescent tag and the second quencher.

52. The composition of claim 50, further comprising a second monomer type include a second quencher having a second quenching efficiency and a third monomer type include a second quencher having a third quenching efficiency, where the first, second and third quenchers are the same or different and the first, second and third quenching efficiencies are the same or different and where the fluorescent tag is quenched during each monomer incorporation of a first, second or third monomer type in a sequence of monomer incorporations due to an interaction between the fluorescent tag and the second or third quencher.

53. The composition of claim 50, further comprising a second monomer type including a second quencher having a second quenching efficiency, a third monomer type including a third quencher having a third quenching efficiency, and a fourth monomer type including a fourth quencher having a fourth quenching efficiency, where the first, second, third and fourth quenchers are the same or different and the first, second, third and fourth quenching efficiencies are the same or different and where the fluorescent tag is quenched during each incorporation of a second, third or fourth monomer type in a sequence of monomer incorporations due to an interaction between the fluorescent tag and the second, third or fourth quencher.

54. The composition of claim 50, wherein the polymerizing agent is a polymerase or reverse transcriptase.

55. The composition of claim 54, wherein the polymerase is selected from the group consisting of Tag DNA polymerase I, T7 DNA polymerase, Sequenase, and the Klenow fragment from E. coli DNA polymerase I and wherein the reverse transcriptase comprises HIV-1 reverse transcriptase.

56. A composition:

comprising a polymerizing agent including a fluorescent tag,

a nucleic acid template, a primer adapted to hybridize to a complementary portion of the template to form a primer-template duplex, and

monomers for the polymerizing agent, where one monomer type includes a first quencher covalently bonded to a terminal phosphate group of one monomer type and where the first quencher has a first quenching efficiency,

where the fluorescent tag is quenched during each monomer incorporation of a first quencher modified monomer in a sequence of monomer incorporations due to an interaction between the fluorescent tag and the first quencher as each first quencher modified monomer is incorporated.

57. The composition of claim 56, further comprising a second monomer type including a second quencher having a second quenching efficiency, where the first and second quenchers are the same or different and the first and second quenching efficiencies are the same or different and where the fluorescent tag is quenched during each incorporation of a first or second quencher modified monomer in a sequence of monomer incorporations due to an interaction between the fluorescent tag and the first or second quencher.

58. The composition of claim 56, further comprising a second monomer type including a second quencher having a second quenching efficiency and a third monomer type including a second quencher having a third quenching efficiency, where the first, second and third quenchers are the same or different and the first, second and third quenching efficiencies are the same or different and where the fluorescent tag is quenched during each incorporation of a second or third monomer type in a sequence of monomer incorporations due to an interaction between the fluorescent tag and the second or third quencher.

59. The composition of claim 56, further comprising a second monomer type including a second quencher having a second quenching efficiency, a third monomer type including a second quencher having a third quenching efficiency, and a fourth monomer type including a second quencher having a fourth quenching efficiency, where the first, second, third and fourth quenchers are the same or different and the first, second, third and fourth quenching efficiencies are the same or different and where the fluorescent tag is quenched during each incorporation of a second, third or fourth monomer type in a sequence of monomer incorporations due to an interaction between the fluorescent tag and the second, third or fourth quencher.

60. The composition of claim 56, wherein the polymerizing agent is a polymerase or reverse transcriptase.

61. The composition of claim 60, wherein the polymerase is selected from the group consisting of Tag DNA polymerase I, T7 DNA polymerase, Sequenase, and the Klenow fragment from E. coli DNA polymerase I and wherein the reverse transcriptase comprises HIV-1 reverse transcriptase.

62. A composition comprising a polymerizing agent including a fluorescent tag associated with the polymerizing agent or covalently bonded to a site on the polymerase and monomer types of the polymerizing agent, where one monomer type includes a molecular and/or atomic tag that reduces fluorescence from the polymerizing agent fluorescent tag before, during and/or after incorporation of the one monomer type due to the interaction between the fluorescent polymerizing agent tag and the monomer tag.

63. The composition of claim 62, further comprising monomer types for the polymerizing agent, where each monomer type includes a molecular and/or atomic tag and where the monomer tags have different quenching signatures.

64. A composition:

comprising a polymerizing agent including a fluorescent polymerizing agent tag,

a nucleic acid template, a primer adapted to hybridize to a complementary portion of the template to form a primer-template duplex,

at least one monomer type includes a quencher covalently bonded thereto, and

at least one monomer type includes a fluorescent monomer tag covalently bonded thereto,

where the quencher reduces a fluorescent intensity of the fluorescent polymerizing agent tag before, during and/or after each incorporation of a quencher modified monomer in a sequence of monomer incorporations or where a fluorescent property of the fluorescent polymerizing agent and/or the fluorescent monomer tag changes before, during and/or after each incorporation of a fluorescent tagged monomer in the sequence of monomer incorporations.

65. The composition of claim 64, wherein the polymerizing agent is a polymerase or reverse transcriptase.

66. The composition of claim 661, wherein the polymerase is selected from the group consisting of Tag DNA polymerase I, T7 DNA polymerase, Sequenase, and the Klenow fragment from E. coli DNA polymerase I and wherein the reverse transcriptase comprises HIV-1 reverse transcriptase.

67. A composition:

comprising a polymerizing agent including a fluorescent tag,

a nucleic acid template, a primer adapted to hybridize to a complementary portion of the template to form a primer-template duplex,

monomer types for the polymerizing agent, where each monomer type includes a quencher or a fluorophore covalently bonded thereto, where the quenchers are the same or different and the fluorophores are the same or different, and

where the quenchers reduce a fluorescent intensity of the fluorescent tag before, during and/or after each incorporation of a quencher modified monomer in a sequence of monomer incorporations or where a fluorescent property of the fluorescent tag and/or the fluorophore changes before, during and/or after each incorporation of a fluorophore modified monomer in the sequence of monomer incorporations.

68. The composition of claim 67, wherein the polymerizing agent is a polymerase or reverse transcriptase.

69. The composition of claim 68, wherein the polymerase is selected from the group consisting of Taq DNA polymerase I, T7 DNA polymerase, Sequenase, and the Klenow fragment from E. coli DNA polymerase I and wherein the reverse transcriptase comprises HIV-1 reverse transcriptase.

70. A method comprising the steps of:

providing a composition comprising a polymerizing agent including a fluorescent tag,

a nucleic acid template, a primer adapted to hybridize to a complementary portion of the template to form a primer-template duplex,

monomer types for the polymerizing agent, where each monomer type includes a quencher or a fluorophore covalently bonded thereto, where the quenchers are the same or different and the fluorophores are the same or different,

detecting a reduction in fluorescent intensity due to the quenchers interacting with the fluorescent tag before, during and/or after each incorporation of a quencher modified monomer in a sequence of monomer incorporations or detecting a change in a fluorescent property of the fluorescent tag and/or the fluorophore before, during and/or after each incorporation of a fluorophore modified monomer in the sequence of monomer incorporations, and

converting the reductions or changes in an identity of monomer incorporations corresponding to a complementary identity of nucleotides of the template.

71. The method of claim 70, wherein the polymerizing agent is a polymerase or reverse transcriptase.

72. The method of claim 71, wherein the polymerase is selected from the group consisting of Taq DNA polymerase I, T7 DNA polymerase, Sequenase, and the Klenow fragment from E. coli DNA polymerase I and wherein the reverse transcriptase comprises HIV-1 reverse transcriptase.