Methods for DNA sequencing with thermus aquaticus DNA polymerase

• US 5,075,216 A
• Filed: 09/23/1988
• Issued: 12/24/1991
• Est. Priority Date: 09/23/1988
• Status: Expired due to Term

• Current Assignee: Roche Molecular Systems (Roche Holding AG)
• Original Assignee: Cetus Corporation (Bayer AG)
• Inventors: Brow, Mary Ann D., Myambo, Kenneth B., Gelfand, David H., Innis, Michael A.
• Primary Examiner(s): Wax, Robert A.
• Assistant Examiner(s): Marschel, Ardin H.
• Application Number:
  US07/249,367
• Time in Patent Office: 1,187 Days
• Field of Search: 435/6, 435/91, 435/810, 436/501, 436/808, 536/27, 935/16, 935/17, 935/18, 935/78, 935/88
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/701 Specific hybridization probes
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• CPC Class Codes: Y10S 435/81 Packaged device or kit
• CPC Class Codes: Y10S 436/808 Automated or kit

Abstract

Dideoxynucleotide DNA sequencing methods can be dramatically improved by utilizing the DNA polymerase from Thermus aquaticus to catalyze the primer extension reactions.

Claims

1. In a method for determining a nucleotide sequence for a nucleic acid segment by a dideoxynucleoside- 5'"'"'-triphosphate chain termination procedure, wherein said sequence is determined by extending an oligonucleotide primer in a templatedependent manner in the presence of an agent for polymerization, four dideoxynucleoside- 5'"'"'-triphosphates (dNTPs), and a dideoxynucleoside- 5'"'"'-triphosphate (ddNTP), the improvement comprising extending said primer in the presence of the agent for polymerization that is Thermus aquaticus DNA polymerase.

2. The method of claim 1, wherein said primer is labeled.

3. The method of claim 1, wherein one of the four dNTPs or ddNTP is labeled.

4. The method of claim 1, wherein the four dideoxynucleoside-5'"'"'-triphosphates are dATP, dCTP, dGTP, and TTP.

5. The method of claim 1, wherein the four dideoxynucleoside- 5'"'"'-triphosphates are dATP, dCTP, c⁷ dGTP, and TTP.

6. The method of claim 1, wherein the four dideoxynucleoside- 5'"'"'-triphosphates are dATP, dCTP, dITP, and TTP.

7. The method of claim 1, wherein said nucleic acid segment was produced by an asymmertric polymerase chain reaction.

8. The method of claim 1, wherein said nucleic acid segment was produced by an asymmetric polymerase chain reaction.

9. The method of claim 1, wherein no KCl is present in the reaction mixture.

10. The method of claim 1, wherein the DNA polymerase is present in up to a 2.5 -fold molar excess over the nucleic acid segment.

11. The method of claim 2, wherein said primer is labeled with ³² P, ³⁵ S, or a fluorescent molecule.

12. The method of claim 3, wherein said dNTP or ddNTP is labeled with ³⁵ S, ³² P, or a fluorescent molecule.

13. The method of claim 3, wherein said extension reaction is carried out first at a low temperature and in the presence of three unlabeled dNTPs and one labeled dNTP each present at a concentration of less than 1 μ

M and then at higher temperatures in higher concentrations of the unlabeled dNTPs.

14. The method of claim 4, wherein said dideoxynucleoside- 5'"'"'-triphosphate is ddATP.

15. The method of claim 4, wherein said dideoxynucleoside- 5'"'"'-triphosphate is ddCTP.

16. The method of claim 4, wherein said dideoxynucleoside- 5'"'"'-triphosphate is ddGTP.

17. The method of claim 4, wherein said dideoxynucleoside- 5'"'"'-triphosphate is ddTTP.

18. The method of claim 4, wherein c⁷ dGTP is also present during primer extension.

19. The method of claim 4, wherein each dNTP is present at a concentration of 5 μ

M to 30 μ

M.

20. The method of claim 13, wherein the concentration of said labeled dNTP is 0.5 μ

M and the concentration of each unlabeled dNTP is 1.0 μ

M during said low temperature extension reaction.

21. The method of claim 14, wherein the dATP:

ddATP ratio is 1;

32.

22. The method of claim 15, wherein the dCTP:

ddCTP ratio is 1;

16.

23. The method of claim 16, wherein the dGTP:

ddGTP ratio is 1;

6.

24. The method of claim 17, wherein the TTP:

ddTTP ratio is 1;

48.

25. The method of claim 19, wherein the concentration of each dNTP is 10 μ

M.

26. A kit for determining a nucleotide sequence for a nucleic acid segment by a dideoxynucleoside-5'"'"'-triphosphate chain termination procedure, which kit comprises

(a) a primer for extending, in a template-dependent manner, a nucleic acid comprising a sequence complementary to said nucleic acid segment;

(b) four deoxyribonucleoside-5'"'"'-triphosphates (dNTPs);

(c) four dideoxyribonucleoside-5'"'"'-triphosphates (ddNTPs); and

(d) Taq polymerase.

27. The kit of claim 26, wherein said dNTPs are dGTP, dATP, TTP, and dCTP, and said ddNTPs are ddGTP, ddATP, ddTTP, and ddCTP.

28. The kit of claim 26 that comprises c⁷ dGTP.

29. The kit of claim 26, wherein said dNTPs are described as a set of four dNTPs selected from the group consisting of:

dGTP, dATP, dCTP, and TTP;

c⁷ dGTP, dATP, dCTP, and TTP; and

dITP, dATP, dCTP, and TTP.

30. The kit of claim 26, wherein said primer hybridizes to an M13 cloning vector and is suitable for sequencing cloned inserts in M13 and/or pUC-based vectors.

31. The kit of claim 27, that comprises:

(i) a G-termination mix comprising dGTP, dATP, TTP, dCTP, and ddGTP;

(ii) a A-termination mix comprising dGTP, dATP, TTP, dCTP, and ddATP;

(iii) a T-termination mix comprising dGTP, dATP, TTP, dCTP, and ddTTP; and

(iv) a C-termination mix comprising dGTP, dATP, TTP, dCTP, and ddCTP.