Method for the uncoupled, direct, exponential amplification and sequence of DNA molecules with the addition of a second thermostable DNA polymerase and its application

• US 6,225,092 B1
• Filed: 12/16/1997
• Issued: 05/01/2001
• 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: Kilger, Christian, Paabo, Svante
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US08/991,184
• Time in Patent Office: 1,232 Days
• Field of Search: 435/91.2, 435/810, 435/91.1
• US Class Current: 435/91.2
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2527/101 Temperature
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• CPC Class Codes: C12Q 2535/113 Cycle sequencing
• CPC Class Codes: C12Q 2549/101 Hot start
• CPC Class Codes: Y10S 435/81 Packaged device or kit

Abstract

Method for sequencing a nucleic acid molecule in a thermocycling reaction which initially comprises a nucleic acid molecule, a first primer, a second primer, a reaction buffer, a first thermostable DNA polymerase, (optionally) a thermostable pyrophosphatase, deoxynucleotides or derivatives thereof and a dideoxynucleotide or a derivative thereof and which is characterized in that the thermocycling reaction additionally contains a second thermostable DNA polymerase which, in comparison to the said first thermostable DNA polymerase, has a reduced ability to incorporate dideoxynucleotides as well as the use of the said method.

Claims

1. A method for sequencing at least a portion of a nucleic acid molecule involving simultaneously amplifying the nucleic acid molecule and generating full length and truncated copies of the nucleic acid molecule for sequencing, comprising the steps of

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

said nucleic acid molecule,

a buffer solution,

a first primer which is able to hybridize with a strand of said nucleic acid molecule,

a second primer which is able to hybridize with a strand of said nucleic acid molecule 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,

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

at least two thermostable DNA polymerases, wherein said at least two thermostable DNA polymerases are at least a first thermostable DNA polymerase and a second thermostable DNA polymerase, which second thermostable DNA polymerase has a reduced ability to incorporate said dideoxynucleotide or another terminating nucleotide compared with said first thermostable DNA polymerase, wherein a ratio of the amount, in unit, of said second thermostable DNA polymerase to the amount, in unit, of said first thermostable DNA polymerase is 1:

X, wherein X is at least 16,

to generate full-length and truncated copies of said nucleic acid molecule, wherein the full-length copies have a length equal to that of at least a portion of said nucleic acid molecule spanning the binding sites of the first and second primers;

(b) making a sequence ladder using at least said truncated copies; and

thereafter

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

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 the thermocycling reaction is carried out from about 18 to about 50 cycles.

4. The method of claim 1, wherein said first thermostable DNA polymerase has a reduced discrimination, compared with wild-type Taq DNA polymerase, against said dideoxynucleotide or another terminating nucleotide relative to deoxynucleotides or deoxynucleotide derivatives.

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

-3′

exonuclease activity and having a Tabor-Richardson mutation.

6. The method of claim 5, wherein said first thermostable DNA polymerase is AmplitaqFS™

, Taquenase™

, or Thermo Sequenase™

.

7. The method of claim 6, wherein said first thermostable DNA polymerase is Thermo Sequenase™

.

8. The method of claim 1, wherein said second thermostable DNA polymerase is Taq DNA polymerase, Tth DNA polymerase, or Klentaq (Taq DNA polymerase)(-exo5′

-3′

).

9. The method of claim 8, wherein said second thermostable DNA polymerase is Taq DNA polymerase.

10. The method of claim 7, wherein said first thermostable DNA polymerase is Thermo Sequenase™

, and said second thermostable DNA polymerase is Taq DNA polymerase.

11. The method of claim 8, wherein said second thermostable DNA polymerase is Tth DNA polymerase, and wherein step (a) is carried out in the presence of MnCl₂ or Mn acetate.

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

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

1.

14. The method of claim 13, wherein said ratio is between about 2:

1 and about 3;

1.

15. The method of claim 14, wherein said ratio is 2:

1.

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

17. The method of claim 1, wherein said annealing and synthesis of the thermocycling reaction is carried out at a temperature of at least 55°

C.

18. The method of claim 1, wherein said nucleic acid molecule in said mixture is a human single-copy DNA.

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

20. The method of claim 1, wherein at least one of the first and second primers has a length that, in combination with a high annealing temperature, prevents annealing to unspecific DNA fragments during the heat denaturation of the thermocycling reaction.

21. The method of claim 20, wherein said length is at least 18 nucleotides.

22. The method of claim 1, wherein said nucleic acid molecule in said mixture is obtained from a body fluid, hairs, a cell, cells or fractions thereof, a tissue or fractions thereof, cell cultures or fractions thereof, bacteria or viruses.

23. The method of claim 1, wherein said nucleic acid molecule in said mixture is total genomic DNA.

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

25. The method of claim 1, wherein said nucleic acid molecule in said mixture is human genomic DNA.

26. The method of claim 1, wherein said nucleic acid molecule in said mixture is purified genomic DNA.

27. The method of claim 1, wherein said nucleic acid molecule in said mixture is unpurified plasmid DNA from bacteria.

28. The method of claim 23, wherein said total genomic DNA has a length of at least 2 kb.

29. The method of claim 1, wherein said nucleic acid molecule in said mixture comprises unpurified cloned DNA.

30. The method of claim 1, wherein said nucleic acid molecule in said mixture comprises purified cloned DNA.

31. The method of claim 1, wherein said nucleic acid molecule in said mixture is a single-copy DNA.

32. The method of claim 31, wherein said nucleic acid molecule in said mixture is at least about 60 ng of a single-copy DNA.

33. The method of claim 23, wherein said nucleic acid molecule in said mixture is at least about 60 ng of total genomic DNA.

34. The method of claim 1, wherein said annealing and synthesis of the thermocycling reaction is carried out at a temperature of at least 66°

C.

35. The method of claim 1, wherein said annealing and synthesis of the thermocycling reaction is carried out at a temperature of at least 68°

C.

36. The method of claim 1, wherein the molar ratio of said deoxynucleotides or deoxynucleotide derivatives to said at least one dideoxynucleotide or another terminating nucleotide is between 100:

1 and 1000;

1.

37. The method of claim 36, wherein the molar ratio of said deoxynucleotides or deoxynucleotide derivatives to said at least one dideoxynucleotide or another terminating nucleotide is between 300:

1 and 600;

1.

38. The method of claim 1, wherein said deoxynucleotides or deoxynucleotide derivatives are present at a concentration of about 300 μ

M to 2 mM.

39. The method of claim 1, wherein said at least one dideoxynucleotide or another terminating nucleotide is present at a concentration of about 1 to 5 μ

M.

40. The method of claim 1, wherein the length of the nucleic acid molecule in said mixture is at least 500 nucleotides between the 3′

ends of the first and second primers.

41. The method of claim 4, wherein said nucleic acid molecule in said mixture comprises RNA and said second thermostable DNA polymerase has reverse transcriptase activity.

42. The method of claim 41, wherein said second thermostable DNA polymerase is Taq DNA polymerase.

43. The method of claim 41, wherein said second thermostable DNA polymerase is Tth DNA polymerase.

59. The method of claim 1, wherein said ratio of the amount of the second thermostable DNA polymerase to the amount of the first thermostable DNA polymerase ranges from 1:

320 to 1;

16.

60. The method of claim 59, wherein said ratio of the amount of the second thermostable DNA polymerase to the amount of the first thermostable DNA polymerase ranges from 1:

128 to 1;

32.

44. A kit for sequencing a nucleic acid molecule, 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; and

at least two thermostable DNA polymerases, wherein said at least two thermostable DNA polymerases are at least a first thermostable DNA polymerase and a second thermostable DNA polymerase, which second thermostable DNA polymerase has a reduced ability to incorporate said dideoxynucleotide or another terminating nucleotide in comparison to said first thermostable DNA polymerase, wherein a ratio of the amount, in unit, of said second thermostable DNA polymerase to the amount, in unit, of said first thermostable DNA polymerase is 1;

X, wherein X is at least 16.

45. The kit of claim 44, wherein said first thermostable DNA polymerase has a reduced discrimination, compared with wild-type Taq DNA polymerase, against said dideoxynucleotide or another terminating nucleotide relative to deoxynucleotides or deoxynucleotide derivatives.

46. The kit of claim 45, wherein said first thermostable DNA polymerase is a Taq DNA polymerase lacking 5′

-3′

exonuclease activity and having a Tabor-Richardson mutation.

47. The kit of claim 46, wherein said first thermostable DNA polymerase is AmplitaqFS™

, Taquenase™

, or ThermoSequenase™

.

48. The kit of claim 47, wherein said first thermostable DNA polymerase is ThermoSequenase™

.

49. The kit of claim 45, wherein said second thermostable DNA polymerase is Taq DNA polymerase, Tth DNA polymerase, or Klentaq (Taq DNA polymerase) (-exo5′

-3′

).

50. The kit of claim 49, wherein said second thermostable DNA polymerase is Taq DNA polymerase.

51. The kit of claim 49, further comprising MnCl₂ or Mn acetate, wherein said second thermostable DNA polymerase is Tth DNA polymerase.

52. The kit of claim 45, further comprising a first primer which is able to hybridize with a strand of said nucleic acid molecule, and a second primer which is able to hybridize with a strand of said nucleic acid molecule complementary to the strand with which the first primer is able to hybridize, wherein the ratio of said first primer to said second primer is not equal to 1:

1.

53. The kit of claim 52, wherein said ratio is between about 2:

1 and about 3;

1.

54. The kit of claim 53, wherein said ratio is 2:

1.

55. The kit of claim 44, further comprising at least one thermostable pyrophosphatase.

56. The kit of claim 44, wherein said second thermostable DNA polymerase has reverse transcriptase activity.

57. The kit of claim 56, wherein said second thermostable DNA polymerase is Taq DNA polymerase.

58. The kit of claim 56, wherein said second thermostable DNA polymerase is Tth DNA polymerase.

61. The kit of claim 44, wherein said ratio of the amount of the second thermostable DNA polymerase to the amount of the first thermostable DNA polymerase ranges from 1:

320 to 1;

16.

62. The kit of claim 61, wherein said ratio of the amount of the second thermostable DNA polymerase to the amount of the first thermostable DNA polymerase ranges from 1:

128 to 1;

32.