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

• US 6,107,032 A
• Filed: 12/16/1997
• Issued: 08/22/2000
• 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): Sisson, Bradley
• Assistant Examiner(s): Siew, Jeffrey
• Application Number:
  US08/991,347
• Time in Patent Office: 980 Days
• Field of Search: 435/6, 435/91.2, 536/24.33
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2527/143 Concentration of primer or probe
• 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.

Claims

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

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

said DNA molecule,

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

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,

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 antibody against said thermostable DNA polymerase, which polymerase-inhibiting antibody 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,

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

(b) generating a sequence ladder by separating at least the truncated copies of said DNA molecule; and

thereafter

(c) reading the sequence ladder to obtain the sequence of said at least a portion of said DNA 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 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 and having a Tabor-Richardson mutation or a functional derivative thereof.

5. The method of claim 4, wherein said thermostable DNA polymerase is AMPLITAQFS, TAQUENASE, THEROSEQUENASE or functional derivatives thereof.

6. The method of claim 5, wherein said thermostable DNA polymerase is THERMOSEQUENASE or a functional derivative thereof.

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 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 is between 100:

1 and 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 is between 300:

1 and 600;

1.

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

M and 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 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 DNA molecule 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, or bacteriophages.

21. The method of claim 1, wherein said DNA molecule is in a complex mixture of nucleic acids.

22. The method of claim 1, wherein said DNA molecule is a single-copy DNA.

23. The method of claim 1, wherein said DNA molecule in mixture A is mitochondrial DNA.

24. The method of claim 1, wherein said DNA molecule in mixture A is unpurified plasmid DNA from bacteria.

25. The method of claim 1, wherein said DNA molecule in mixture A is unpurified single-stranded or double-stranded DNA from bacteriophages.

26. The method of claim 21, wherein said complex mixture of nucleic acids in mixture A is total genomic DNA.

27. The method of claim 26, wherein said total genomic DNA is unpurified.

28. A kit for sequencing at least a portion of 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;

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

29. The kit of claim 28, wherein said deoxynucleotide derivatives are thionucleotides, 7-deaza-2'"'"'-dGTP, 7-deaza-2'"'"'-dATP or deoxyinosine triphosphate.

30. The kit of claim 28, wherein said another terminating nucleotide is a 3'"'"'-aminonucleotide or a nucleotide having an ester group at the 3'"'"' position.

31. The kit of claim 28, 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.

32. The kit of claim 28, wherein said thermostable DNA polymerase is AMPLITAQFS, TAQUENASE, THERMOSEQUENASE or functional derivatives thereof.

33. The kit of claim 32, wherein said thermostable DNA polymerase is THERMOSEQUENASE or functional derivatives thereof.

34. The kit of claim 28, further comprising at least one thermostable pyrophosphatase.

35. The kit of claim 28, wherein the molar ratio of said deoxynucleotides and deoxynucleotide derivatives to said dideoxynucleotide and another terminating nucleotide is between 100:

1 and 1000;

1.

36. The kit of claim 35, wherein the molar ratio of said deoxynucleotides and deoxynucleotide derivatives to said dideoxynucleotide and another terminating nucleotide is between 300:

1 and 600;

1.

37. The kit of claim 28, further comprising first and second primers, wherein

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

(2) said second primer is able to hybridize with a strand of said DNA molecule complementary to the strand said first primer is able to hybridize with,

wherein at least one of the first and second primers is labelled.

38. The kit of claim 37, wherein the molar ratio of said first primer to said second primer is different from 1:

1.

39. The kit of claim 38, wherein the molar ratio of said first primer to said second primer is about 2:

1 to about 3;

1.

40. The kit of claim 39, wherein the molar ratio of said first primer to said second primer is 2:

1.

41. The kit of claim 37, wherein the first and second primers are differently labelled.

42. The kit of claim 37, wherein the first and second primers independently have a length of at least 25 nucleotides.