Method for the direct, exponential amplification and sequencing of DNA molecules and its application

• US 6,605,428 B2
• Filed: 06/24/1999
• Issued: 08/12/2003
• Est. Priority Date: 12/20/1996
• Status: Expired due to Fees

• Current Assignee: Roche Diagnostics GmbH (Roche Holding AG)
• Original Assignee: Roche Diagnostics GmbH (Roche Holding AG)
• Inventors: Paabo, Svante, Kilger, Christian
• Primary Examiner(s): Siew, Jeffrey
• Application Number:
  US09/339,104
• Publication Number:
  US 20030134276A1
• Time in Patent Office: 1,510 Days
• Field of Search: 435/6, 435/89, 435/91.1, 435/91.2, 435/91.21, 435/91.31, 435/91.32, 435/91.33, 435/91.4, 435/91.41, 435/91.42, 435/91.5, 435/91.51, 435/183, 435/193, 326/22.1, 326/23.1, 326/23.2, 326/23.4, 326/23.5, 326/23.72, 326/24.3, 326/24.31, 326/24.32, 326/24.33, 326/24.5, 326/25.3, 326/25.32, 530/350
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2535/113 Cycle sequencing

Abstract

A method is described for the direct, exponential amplification and sequencing (“DEXAS”) of a DNA molecule from a complex mixture of nucleic acids, wherein truncated DNA molecules as well as DNA molecules of full length are synthesized simultaneously and exponentially between two positions on the said DNA molecule, which initially contains a DNA molecule in a thermocycling reaction, a first primer, a second primer, a reaction buffer, a thermostable DNA polymerase, a thermostable pyrophosphatase (optionally), deoxynucleotides or derivatives thereof and a dideoxynucleotide or derivatives thereof. In a preferred embodiment of the method of the invention, direct sequencing of RNA can be performed using one polymerase having a Tabor-Richardson mutation, or a functional derivative thereof, and reverse transcriptase activity. In a more preferred embodiment of the method of the invention, direct sequencing of RNA can be performed in one step, in one vessel.

Claims

1. A method for sequencing at least a portion of a RNA involving converting said RNA into a DNA and simultaneously amplifying said DNA and generating truncated copies of said DNA for sequencing, comprising the steps of

(a) subjecting a mixture A to conversion of said RNA to said DNA and, in a single step, to DNA amplification and generation of truncated copies of said DNA by subjecting the mixture A to a thermocycling reaction, the thermocycling reaction comprises heat denaturation, annealing and synthesis, wherein said mixture A comprises said RNA,

a first primer which is able to hybridize with a strand of said DNA, a second primer which is able to hybridize with a strand of DNA complementary to the strand with which the first primer is able to hybridize, wherein at least one of the first and second primers is labelled,

a reaction buffer,

deoxynucleotides or deoxynucleotide derivatives, wherein said deoxynucleotide derivatives are able to be incorporated by a thermostable DNA polymerase into growing DNA molecules in place of one of dATP, dGTP, dTTP or dCTP,

at least one dideoxynucleotide or another terminating nucleotide, and

a thermostable DNA polymerase having a reduced, compared to wild-type Taq DNA polymerase, discrimination against the incorporation of dideoxynucleotides relative to deoxynucleotides, wherein said thermostable DNA polymerase has (1) a Tabor-Richardson mutation, or a functional derivative thereof, and (2) reverse transcriptase activity, to simultaneously make full-length and truncated copies of said DNA, wherein the full-length copies have a length equal to that of at least a portion of said DNA spanning the binding sites of the first and second primers;

(b) separating at least the truncated copies of said DNA to obtain a sequence ladder; and

thereafter

(c) reading the sequence ladder to obtain the sequence of said at least a portion of said RNA.

2. The method of claim 1, wherein the deoxynucleotide derivatives are thionucleotides, 7-deaza-2′

-dGTP, 7-deaza-2′

-dATP or deoxyinosine triphosphate.

3. The method of claim 1, wherein said another terminating nucleotide is 3′

-aminonucleotide or a nucleotide having an ester group at the 3′

position.

4. The method of claim 1, wherein said thermostable DNA polymerase is a Taq DNA polymerase lacking 5′

-3′

exonuclease activity.

5. The method of claim 1, wherein said thermostable DNA polymerase is Taq DNA polymerase, Tth DNA polymerase, Tfl DNA polymerase or a DNA polymerase from Carboxydothermus hydrogenoformans having a Tabor-Richardson mutation, or a functional derivative of the Tabor-Richardson mutation.

6. The method of claim 1, wherein said thermostable DNA polymerase is Tth DNA polymerase having a Tabor-Richardson mutation, or a functional derivative of the Tabor-Richardson mutation.

7. The method of claim 1, wherein the thermocycling reaction in step (a) is carried out without interruption in a single container, vessel or tube.

8. The method of claim 1, wherein the initial molar ratio of said first primer to said second primer is not equal to 1:

1 in step (a).

9. The method of claim 8, wherein the initial molar ratio of said first primer to said second primer is about 2:

1 to about 3;

1 in step (a).

10. The method of claim 9, wherein the initial molar ratio of said first primer to said second primer is about 2:

1 in step (a).

11. The method of claim 1, wherein the first and second primers are differently labelled.

12. The method of claim 1, wherein the first and second primers independently have a length of at least 25 nucleotides.

13. The method of claim 1, wherein the initial ratio of said deoxynucleotides and deoxynucleotide derivatives to said dideoxynucleotide and another terminating nucleotide in mixture A is between about 100:

1 and about 1000;

1.

14. The method of claim 13, wherein the initial ratio of said deoxynucleotides and deoxynucleotide derivatives to said dideoxynucleotide and another terminating nucleotide in mixture A is between about 300:

1 and about 600;

1.

15. The method of claim 1, wherein the initial concentrations of said deoxynucleotides or deoxynucleotide derivatives in mixture A are between about 300 μ

M and about 2 mM.

16. The method of claim 1, wherein the initial concentration of said dideoxynucleotide or another terminating nucleotide in mixture A is between about 1 and about 5 μ

M.

17. The method of claim 1, wherein mixture A further comprises at least one thermostable pyrophosphatase.

18. The method of claim 1, wherein the annealing and synthesis of the thermocycling reaction are carried out at a temperature of at least about 66°

C.

19. The method of claim 18, wherein the annealing and synthesis of the thermocycling reaction are carried out at a temperature of at least about 68°

C.

20. The method of claim 1, wherein said RNA in mixture A is obtained from a body fluid, hairs, an individual cell, cells or fractions thereof, a tissue or fractions thereof, a cell culture or fractions thereof, a tissue culture or fractions thereof, a bacteria or a virus.

21. The method of claim 1, wherein said RNA in mixture A is an RNA in a complex mixture of nucleic acids.

22. The method of claim 1, wherein said RNA in mixture A is an RNA mixed with total genomic DNA.

23. The method of claim 22, wherein said total genomic DNA is unpurified.

24. The method of claim 1, wherein said RNA in mixture A is unpurified RNA.

25. The method of claim 1, wherein said RNA in mixture A is RNA from single copy genes.

26. The method of claim 1, wherein mixture A further comprises at least one polymerase-inhibiting agent against said thermostable DNA polymerase, which polymerase-inhibiting agent loses inhibitory ability, thereby allowing said thermostable DNA polymerase to be active, at a temperature which is at least the temperature at which unspecifically hybridized primers separate from a DNA molecule.

27. The method of claim 26, wherein said at least one polymerase-inhibiting agent is a compound containing at least one acid anhydride group per molecule.

28. The method of claim 27, wherein said at least one polymerase-inhibiting agent is citraconic anhydride, cis-aconitic anhydride, phthalic anhydride, succinic anhydride, or maleic anhydride.

29. The method of claim 27, wherein said at least one polymerase-inhibiting agent is a compound having at least two acid anhydride groups per molecule.

30. The method of claim 27, wherein said at least one polymerase-inhibiting agent is pyromellitic dianhydride or naphthalenetetracarboxylic dianhydride.

31. The method of claim 26, wherein said at least one polymerase-inhibiting agent is an antibody against said thermostable DNA polymerase.

32. The method of claim 1, wherein the first and second primers independently have a length of at least 16 nucleotides.

33. The method of claim 1, wherein the annealing and synthesis of the thermocycling reaction are carried out at a temperature of at least about 55°

C.

87. The method of claim 1, wherein said thermostable DNA polymerase is Tth DNA polymerase having a Tabor Richardson mutation.

34. A method for sequencing at least a portion of a DNA involving simultaneously amplifying said DNA and generating truncated copies of said DNA for sequencing, comprising the steps of

(a) subjecting a mixture, in a single step, to DNA amplification and generation of truncated copies of said DNA by subjecting the mixture to a thermocycling reaction, the thermocycling reaction comprises heat denaturation, annealing and synthesis, wherein said mixture comprises said DNA,

a first primer which is able to hybridize with a strand of said DNA,

a second primer which is able to hybridize with a strand of DNA complementary to the strand with which the first primer is able to hybridize, wherein at least one of the first and second primers is labelled,

a reaction buffer,

deoxynucleotides or deoxynucleotide derivatives, wherein said deoxynucleotide derivatives are able to be incorporated by a thermostable DNA polymerase into growing DNA molecule, in place of one of dATP, dGTP, dTTP or dCTP, m

at least one dideoxynucleotide or another terminating nucleotide

a thermostable DNA polymerase having a reduced, compared to wild-type Taq DNA polymerase, discrimination against the incorporation of dideoxynucleotides relative to deoxynucleotides, and

at least one polymerase-inhibiting agent against said thermostable DNA polymerase, which polymerase-inhibiting agent loses inhibitory ability, thereby allowing said thermostable DNA polymerase to be active, at a temperature which is at least the temperature at which unspecifically hydridized primers separate from a DNA molecule, to simultaneously make full-length and truncated copies of said DNA, wherein the full-length copies have a length equal to that of at least a portion of said DNA spanning the binding sites of the first and second primers;

(b) separating at least the truncated copies of said DNA to obtain a sequence ladder; and

thereafter

(c) reading the sequence ladder to obtain the sequence of said at least a portion of said DNA.

35. The method of claim 34, wherein the deoxynucleotide derivatives are thionucleotides, 7-deaza-2′

-dGTP, 7-deaza-2′

-dATP or deoxyinosine triphosphate.

36. The method of claim 34, wherein said another terminating nucleotide is 3′

-aminonucleotide or a nucleotide having an ester group at the 3′

position.

37. The method of claim 34, wherein said thermostable DNA polymerase is a Taq DNA polymerase lacking 5′

-3′

exonuclease activity and having a Tabor-Richardson mutation or a functional derivative thereof.

38. The method of claim 37, wherein said thermostable DNA polymerase is AMPLITAQFS, TAQUENASE, THERMOSEQUENASE, or functional derivatives thereof.

39. The method of claim 38, wherein said thermostable DNA polymerase is THERMOSEQUENASE or a functional derivative thereof.

40. The method of claim 34, wherein the thermocycling reaction in step (a) is carried out without interruption in a single container, vessel or tube.

41. The method of claim 34, wherein the initial molar ratio of said first primer to said second primer is not equal to 1:

1 in step (a).

42. The method of claim 41, wherein the initial molar ratio of said first primer to said second primer is 2:

1 to 3;

1 in step (a).

43. The method of claim 42, wherein the initial molar ratio of said first primer to said second primer is 2:

1 in step (a).

44. The method of claim 34, wherein the first and second primers are differently labelled.

45. The method of claim 34, wherein the first and second primers independently have a length of at least 25 nucleotides.

46. The method of claim 34, wherein the initial ratio of said deoxynucleotides and deoxynucleotide derivatives to said dideoxynucleotide and another terminating nucleotide in mixture A is between 100:

1 and 1000;

1.

47. The method of claim 46, wherein the initial ratio of said deoxynucleotides and deoxynucleotide derivatives to said dideoxynucleotide and another terminating nucleotide in mixture A is between 300:

1 and 600;

1.

48. The method of claim 34, wherein the initial concentrations of said deoxynucleotides or deoxynucleotide derivatives in said mixture are between 300 μ

M and 2 mM.

49. The method of claim 48, wherein the initial concentration of said dideoxynucleotide or another terminating nucleotide in said mixture is between 1 and 5 μ

M.

50. The method of claim 34, wherein said mixture further comprises at least one thermostable pyrophosphatase.

51. The method of claim 34, wherein the annealing and synthesis of the thermocycling reaction are carried out at a temperature of at least 66°

C.

52. The method of claim 34, wherein the annealing and synthesis of the thermocycling reaction are carried out at a temperature of at least about 68°

C.

53. The method of claim 34, wherein said DNA in said mixture is obtained from a body fluid, hairs, an individual cell, cells or fractions thereof, a tissue or fractions thereof, a cell culture or fractions thereof, a tissue culture or fractions thereof, a bacteria or a virus.

54. The method of claim 34, wherein said DNA in said mixture is a DNA in a complex mixture of nucleic acids.

55. The method of claim 54, wherein said DNA in said mixture is a DNA mixed with total genomic DNA.

56. The method of claim 55, wherein said total genomic DNA is unpurified.

57. The method of claim 34, wherein said DNA in said mixture is unpurified DNA.

58. The method of claim 34, wherein said DNA in said mixture is a DNA from single copy genes.

59. The method of claim 34, wherein said at least one polymerase-inhibiting agent is a compound containing at least one acid anhydride group per molecule.

60. The method of claim 59, wherein said at least one polymerase-inhibiting agent is citraconic anhydride, cis-aconitic anhydride, phthalic anhydride, succinic anhydride, or maleic anhydride.

61. The method of claim 60, wherein said at least one polymerase-inhibiting agent is a compound having at least two acid anhydride groups per molecule.

62. The method of claim 61, wherein said at least one polymerase-inhibiting agent is pyromellitic dianhydride or naphthalenetetracarboxylic dianhydride.

63. The method of claim 60, wherein said at least one polymerase-inhibiting agent is citraconic anhydride or cis-aconitic anhydride.

85. The method of claim 34, wherein the first and second primers independently have a length of at least 16 nucleotides.

86. The method of claim 34, wherein the annealing and synthesis of the thermocycling reaction are carried out at a temperature of at least about 55°

C.

64. A kit for sequencing at least a portion of a DNA comprising deoxynucleotides or deoxynucleotide derivatives, which deoxynucleotide derivatives are able to be incorporated by a thermostable DNA polymerase into growing DNA molecules in place of one of dATP, dGTP, dTTP or dCTP;

at least one dideoxynucleotide or another terminating nucleotide;

a thermostable DNA polymerase having a reduced, compared with wild-type Taq polymerase, discrimination against the incorporation of dideoxynucleotides relative to deoxynucleotides, and

at least one polymerase-inhibiting agent against said thermostable DNA polymerase, which polymerase-inhibiting agent loses inhibitory ability, thereby allowing said thermostable DNA polymerase to be active, at a temperature which is at least the temperature at which unspecifically hydridized primers separate from a DNA molecule.

65. The kit of claim 64, wherein said deoxynucleotide derivatives are thionucleotides, 7-deaza-2′

-dGTP, 7-deaza-2′

-dATP or deoxyinosine triphosphate.

66. The kit of claim 64, wherein said another terminating nucleotide is a 3′

-aminonucleotide or a nucleotide having an ester group at the 3′

position.

67. The kit of claim 64, wherein said thermostable DNA polymerase is a Taq DNA polymerase lacking 5′

-3′

exonuclease activity and having a Tabor-Richardson mutation or a functional derivative of the Tabor-Richardson mutation.

68. The kit of claim 64, wherein said thermostable DNA polymerase is AMPLITAQFS, TAQUENASE, THERMOSEQUENASE, or functional derivatives thereof.

69. The kit of claim 68, wherein said thermostable DNA polymerase is THERMOSEQUENASE, or a functional derivative thereof.

70. The kit of claim 64, further comprising at least one thermostable pyrophosphatase.

71. The kit of claim 64, wherein the molar ratio of said deoxynucleotides and deoxynucleotide derivatives to said dideoxynucleotide and another terminating nucleotide is between 100:

1 and 1000;

1.

72. The kit of claim 71, wherein the molar ratio of said deoxynucleotides and deoxynucleotide derivatives to said dideoxynucleotide and another terminating nucleotide is between 300:

1 and 600;

1.

73. The kit of claim 64, wherein said at least one polymerase-inhibiting agent is a compound containing at least one acid anhydride group per molecule.

74. The kit of claim 73, wherein said at least one polymerase-inhibiting agent is citraconic anhydride, cis-aconitic anhydride, butyric anhydride, acetic propionic anhydride, phthalic anhydride, succinic anhydride, maleic anhydride or phthalic anhydride.

75. The kit of claim 64, wherein said at least one polymerase-inhibiting agent is a compound having at least two acid anhydride groups per molecule.

76. The kit of claim 75, wherein said at least one polymerase-inhibiting agent is pyromellitic dianhydride or naphthalenetetracarboxylic dianhydride.

77. The kit of claim 74, wherein said at least one polymerase-inhibiting agent is citraconic anhydride or cis-aconitic anhydride.

78. The kit of claim 64, further comprising first and second primers, wherein

(1) said first primer is able to hybridize with a strand of a DNA to be sequenced, and

(2) said second primer is able to hybridize with a strand of a DNA complementary to the strand with which said first primer is able to hydridize, wherein at least one of the first and second primers is labelled.

79. The kit of claim 78, wherein the molar ratio of said first primer to said second primer is different from 1:

1.

80. The kit of claim 79, wherein the molar ratio of said first primer to said second primer is 2:

1 to 3;

1.

81. The kit of claim 80, wherein the molar ratio of said first primer to said second primer is 2:

1.

82. The kit of claim 78, wherein the first and second primers are differently labelled.

83. The kit of claim 78, wherein the first and second primers independently have a length of at least 16 nucleotides.

84. The kit of claim 83, wherein the first and second primers independently have a length of at least 25 nucleotides.