Method of using a TAQ DNA polymerase without 5'-3'-exonuclease activity

• US 5,108,892 A
• Filed: 08/03/1989
• Issued: 04/28/1992
• Est. Priority Date: 08/03/1989
• Status: Expired due to Term

• Current Assignee: Promega Corporation
• Original Assignee: Promega Corporation
• Inventors: Spurgeon, Sandra L., Burke, Thomas J., Thompson, David V.
• Primary Examiner(s): Wax, Robert A.
• Assistant Examiner(s): Zitomer, Stephanie W.
• Application Number:
  US07/389,745
• Time in Patent Office: 999 Days
• Field of Search: 435/6, 435/91, 435/194, 435/280, 435/243, 436/501, 436/94, 935/77, 935/78
• US Class Current: 435/6.12
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• 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: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

The present invention is directed to a modified Taq DNA polymerase and methods for its use.

Claims

1. A method for determining the nucleotide base sequence of a DNA molecule, comprising:

a) hybridizing a primer to a DNA template molecule to be sequenced;

b) extending the primer with a Taq DNA polymerase having an apparent molecular weight of about 80,000 daltons and substantially no 5'"'"'-3'"'"' exonuclease activity;

c) incorporating a strand terminating nucleotide onto the extended primer under conditions to yield a distinct population of nucleotide fragments beginning on the same nucleotide position for all populations and having a variable terminus at one of the following four nucleotide bases;

A, C, G and T; and

d) separating the synthesized fragments according to their size, whereby at least a part of the nucleotide base sequence of the DNA molecule can be determined.

2. The method of claim 1 wherein the primer is an oligonucleotide primer.

3. The method of claim 1 wherein the determination of the DNA sequence further comprises a set of four different sequencing reactions, wherein each sequencing reaction contains a different strand terminating nucleotide which terminates nucleotide synthesis at a specific nucleotide base wherein each reaction yields a distinct population of nucleotide fragments.

4. The method of claim 3 wherein each sequencing reaction contains the following four deoxyribonucleotide triphosphates:

dATP, dCTP, dGTP, dTTP; and

each sequencing reaction contains a limiting amount of strand terminating nucleotide.

5. The method of claim 4 wherein the strand terminating nucleotide is selected from the following dideoxyribonucleotide triphosphates:

ddATP, ddCTP, ddGTP, and ddTTP.

6. The method of claim 5 further comprising adjusting the concentration of the dideoxyribonucleotide triphosphate depending on the number of nucleotide bases to be sequenced.

7. The method of claim 1 further comprising incorporating a radioactive nucleotide in the elongating DNA strand to detect the sequencing products.

8. The method of claim 7 comprising:

a) hybridizing the primer to a DNA template to be sequenced;

b) extending the primer with the Taq DNA polymerase in four separate reactions, each reaction containing one alpha-³² P-labeled deoxynucleotide triphosphate selected from the group consisting of dATP, dCTP, dGTP, and dTTP;

each reaction further containing unlabeled dATP, dCTP, dGTP, and dTTP;

each reaction further comprising a different one of a chain terminating dideoxyribonucleotide triphosphate selected from the group consisting of ddATP, ddCTP, ddGTP, and ddTTP;

c) resolving the products of the reactions on a gel using high-resolution denaturing polyacrylamide gel electrophoresis; and

d) employing radioactivity of the ³² P incorporated into the reaction products to produce a detectable image of the gel to determine the DNA sequence.

9. The method of claim 1 further comprising incorporating a nonradioactively-labeled nucleotide in the elongating DNA strand to detect the sequencing products.

10. The method of claim 9 wherein the nonradioactively-labeled nucleotide is selected from the group consisting of biotin and fluorescently labeled nucleotides.

11. A method for determining the nucleotide base sequence of a DNA molecule, comprising:

a) hybridizing a primer to a DNA template molecule to be sequenced;

b) extending the primer with a Taq DNA polymerase having an apparent molecular weight of about 80,000 daltons and substantially no 5'"'"'-3'"'"' exonuclease activity;

c) incorporating alpha-thiodeoxynucleotides onto the extending primer with the modified Taq DNA polymerase, wherein the extended primer is selectively degraded to yield a distinct population of nucleotide fragments beginning at the same nucleotide position for all populations and having a variable terminus at one of the following four nucleotide bases;

A, C, G and T; and

d) separating the synthesized fragments according to size, whereby at least a part of the nucleotide base sequence of the DNA molecule can be determined.

12. A method for determining the nucleotide base sequence of a DNA molecule, comprising:

a) hybridizing a primer to a DNA template molecule to be sequenced;

b) extending the primer with a Taq DNA polymerase having an apparent molecular weight of about 80,000 daltons and substantially no 5'"'"'-3'"'"' exonuclease activity;

c) incorporating a strand terminating nucleotide onto the extended primer, wherein the determination of the DNA sequence is accomplished by a set of four different sequencing reactions, wherein each sequencing reaction contains a different strand terminating nucleotide which terminates nucleotide synthesis at a specific nucleotide base wherein each reaction yields a distinct population of nucleotide fragments beginning on the same nucleotide position for all populations, but having a variable terminus at one of the following four nucleotide bases;

A, C, G and T; and

d) separating the synthesized fragments according to their size, whereby at least a part of the nucleotide base sequence of the DNA molecule can be determined.

13. The method of claim 12, wherein the Taq DNA polymerase produces readable DNA sequence data for up to 1000 nucleotide bases before dissociating.

14. The method of claim 12 wherein the Taq DNA polymerase produces readable DNA sequence data for up to 500 nucleotide bases before dissociating.

15. The method of claim 12 wherein the synthesized fragments are separated by denaturing polyacrylamide gel electrophoresis.

16. The method of claim 12 wherein each sequencing reaction contains the following four deoxyribonucleotide triphosphates:

dATP, dCTP, dGTP, dTTP; and

each sequencing reaction contains a limiting amount of a strand terminating nucleotide selected from one of the following dideoxyribonucleotide triphosphates;

ddATP, ddCTP, ddGTP, ddTTP.

17. The method of claim 16 further comprising adjusting the concentration of the dideoxyribonucleotide triphosphates depending on the number of nucleotide bases to be sequenced, wherein the concentrations are optimized for incorporation by the modified Taq DNA polymerase.

18. The method of claim 12 further comprising incorporating a radioactive nucleotide in the elongating DNA strand to detect the sequencing products.

19. The method of claim 12 further comprising incorporating a nonradioactively-labeled nucleotide in the elongating DNA strand to detect the sequencing products.

20. The method of claim 19 wherein the nonradioactively-labeled nucleotide is selected from the group consisting of biotin and fluorescently labeled nucleotides.

21. The method of claim 18 further comprising end-labeling the primer in the presence of an effective end-labeling amount of a radiolabeled nucleotide prior to hybridizing the DNA template.

22. The method of claim 21 the radiolabeled nucleotide is labeled with gamma³⁵ S or gamma ³² P.

23. The method of claim 21 further comprising end-labeling the primer in the presence of an effective end-labeling amount of a nonradiolabeled nucleotide prior to hybridizing the DNA template.

24. The method of claim 12 wherein the DNA template molecule is selected from the group consisting of single-stranded DNA molecules and denatured double-stranded DNA molecules.

25. The method of claim 18 further comprising extension-labeling the primer in the presence of an effective radioactive nucleotide and non-radioactive deoxyribonucleotide triphosphates.

26. In an enzymatic CNA synthesis reaction wherein a primer is hybridized to a template DNA template in a reaction catalyzed by a DNA polymerase, the improvement comprising catalyzing the extension of primer with a Taq DNA polymerase having an apparent molecular weight of about 80,000 daltons and substantially no 5'"'"'-3'"'"' exonuclease activity.

27. A method of amplifying a DNA sequence comprising:

(a) annealing a first and second primer to opposite strands of a double stranded DNA template molecule, and

(b) thermocycling the annealed mixture with a Taq DNA polymerase having an apparent molecular weight of about 80,000 daltons and substantially no 5'"'"'-3'"'"' exonuclease activity.

28. The method of claim 27 wherein the first and second primers are aligned with their 3'"'"' ends facing each other.

29. A thermocycling DNA sequencing method comprising one or more repetitions of the following steps:

a) denaturing the DNA sequence under suitable denaturing conditions to form single-stranded DNA sequence segments;

b) annealing the DNA sequence under conditions sufficient to hybridize a primer to a template of a DNA sequence segment; and

c) replicating the DNA sequence segment by extending the primer with a Taq DNA polymerase having an apparent molecular weight of about 80,000 daltons and substantially no 5'"'"'-3'"'"' exonuclease activity.

30. A method for determining the order in which nucleotide bases are arranged within a length of a nucleic acid strand, comprising:

a) incubating the nucleic acid strand with a nucleotide triphosphate selected from the group consisting of dATP, dCTP, dGTP, dTTP, and a primer for each sequence;

b) incubating the nucleic acid strand at the same time with a Taq DNA polymerase having an apparent molecular weight of about 80,000 daltons and substantially no 5'"'"'-3'"'"' exonuclease activity for an effective time and at an effective temperature to catalyze the nucleotide triphosphate to form primer extension products complementary to each nucleic acid strand.

31. The method of claim 30 wherein the primer is radiolabeled for detection.

32. The method of claim 30 wherein the incorporated nucleotide triphosphate has a radiolabel.

33. The method of claim 32 wherein before hybridizing primer to the template, the sequencing primer is end-labeled.

34. The method of claim 33 wherein the sequencing primer is end-labeled in the presence of T4 polynucleotide kinase and an appropriate gamma-labeled nucleotide.

35. The method of claim 32 wherein the radiolabeled nucleotide is incorporated throughout the length of the newly synthesized nucleic acid strand.

36. The method of claim 30 further comprising incorporating a nonradioactively-labeled nucleotide in the elongating DNA strand to detect the sequencing products.

37. The method of claim 36 wherein the nonradioactively-labeled nucleotide is selected from the group consisting of biotin and fluorescently labeled nucleotides.

38. The method of claim 30 wherein the reaction proceeds at a temperature up to about 96°

C.

39. The method of claim 30 wherein the reaction proceeds at a temperature between 70°

and 80°

C.

40. The method according to claim 30 wherein the nucleic acid strand is denatured double-stranded DNA or single-stranded DNA.

42. The method of claim 40 wherein the labeling nucleotide is selected from the group consisting of radioactive nucleotides and nonradioactive nucleotides.

41. A method for labeling DNA molecules comprising:

a) annealing a primer to a DNA template molecule; and

b) incubating a nucleotide in the annealed mixture of step a) with a Taq DNA polymerase having an apparent molecular weight of about 80,000 daltons and substantially no 5'"'"'-3'"'"' exonuclease activity under effective conditions to incorporate the labeled nucleotide into the DNA molecule.

43. A method for site-directed in vitro mutagenesis within a DNA sequence comprising annealing a primer containing a desired mutation sequence to a DNA template molecule and extending the primer with a Taq DNA polymerase having a molecular weight of about 80,000 daltons and substantially no 5'"'"'-3'"'"' exonuclease activity, such that the primer synthesizes a new DNA sequence containing the mutation.

44. A method for random in vitro mutagenesis within a DNA sequence comprising annealing a primer to the DNA sequence in the presence of a Taq DNA polymerase having a molecular weight of about 80,000 daltons and substantially no 5'"'"'-3'"'"' exonuclease activity, under conditions in which the DNA sequence is randomly mutated as the new DNA strand is synthesized.

45. A kit for DNA sequencing, comprising:

a) Taq DNA polymerase having an apparent molecular weight of about 80,000 daltons and substantially no 5'"'"'-3'"'"' exonuclease activity; and

b) a reagent necessary for sequencing.

46. The kit of claim 45 wherein the reagent is selected from the group consisting of deoxyribonucleotides, chain terminating agents, DNA sequencing primers, a reaction buffer and mixtures thereof.