Compositions and methods utilizing DNA polymerases

• US 20030157483A1
• Filed: 06/29/2001
• Published: 08/21/2003
• Est. Priority Date: 10/29/1999
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Hogrefe, Holly, Sorge, Joseph A., Hansen, Connie Jo
• Granted Patent: US 8,268,605 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B82Y 10/00 Nanotechnology for information processing, storage or transmission, e.g. quantum computing or single electron logic
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12N 9/1276 RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase

Abstract

The invention features a novel isolated Family B DNA polymerase, a Thermococcus polymerase JDF-3, and mutant recombinant forms thereof. Mutant polymerases of the invention are deficient in 3′ to 5′ exonuclease activity and/or exhibit reduced discrimination against non-conventional nucleotides relative to the wild-type form of the polymerase.

Claims

1. A composition for identifying a nucleotide at a given position of a template DNA molecule, said composition comprising a Family B DNA polymerase having reduced discrimination against non-conventional nucleotides and a first primer, wherein said first primer anneals to the immediate 3′

of said nucleotide at the given position of said template DNA molecule.

2. The composition of claim 1, wherein said Family B DNA polymerase is a JDF-3 DNA polymerase.

3. The composition of claim 2, wherein said JDF-3 DNA polymerase has a sequence of SEQ ID NO:

2 and further comprises one or more amino acid mutations at D141, E143, A485, L408 or P410.

4. The composition of claim 3, wherein said JDF-3 DNA polymerase has one or more amino acid mutations selected from the group consisting of:

D141A or D141T, E143A, L408H or L408F, A485T, and P410L.

5. The composition of claim 4, wherein said JDF-3 DNA polymerase comprises four amino acid mutations at D141 A, E 143A, P410L and A485T.

6. The composition of claims 1-5, wherein said Family B DNA polymerase is deficient in 3′

to 5′

exonuclease activity.

7. The composition of claim 1, further comprising at least one chain-terminating nucleotide analog, wherein said chain-terminating nucleotide analog is incorporated into said first primer by said Family B DNA polymerase in a template-dependent manner.

8. The composition of claim 1, wherein at least one chain-terminating nucleotide analog is labeled with a first detectable label.

9. The composition of claim 1, wherein more than one chain-terminating nucleotide analog is labeled, each chain-terminating nucleotide analog being labeled with a different first detectable label.

10. The composition of claim 7, wherein said chain-terminating nucleotide analog is a dideoxynucleotide.

11. The composition of claim 10, wherein said dideoxynucleotide is selected from the group consisting of:

ddATP, ddTTP, ddCTP and ddGTP.

12. The composition of claim 8, wherein said first primer is labeled with a second detectable label.

13. The composition of claim 12, wherein first and second detectable labels generate a signal for identifying said nucleotide at the given position of the template DNA molecule.

14. The composition of claim 1, further comprising a second primer.

15. The composition of claim 14, wherein said first primer is labeled with a second detectable label and said second primer is labeled with a third detectable label, said second and third detectable labels generate a signal for identifying said nucleotide at the given position of the template DNA molecule.

16. The composition of claim 15, wherein said second primer anneals to the immediate 5′

of said nucleotide at the given position of said template DNA molecule.

17. The composition of claim 16, further comprising a DNA ligase.

18. The composition of claim 1, further comprising a reaction buffer for said Family B DNA polymerase.

19. The composition of claim 1, wherein said template DNA molecule is the product of a polymerase chain reaction or a plasmid DNA.

20. The composition of claim 8, 12, or 15, wherein said first or second or third detectable label is one selected from the group consisting of:

a fluorescent label, an isotope, a chemiluminescent label, a quantum dot label, an antigen, or an affinity moiety.

21. The composition of claim 20, wherein said first detectable label is a rhodamine label or a cyanine label.

22. A kit for identifying a nucleotide at a given position of a template DNA molecule, said kitcomprising a Family B DNA polymerase having reduced discrimination against non-conventional nucleotides and a first primer, wherein said first primer anneals to the immediate 3′

of said nucleotide at the given position of said template DNA molecule.

23. The kit of claim 22, wherein said Family B DNA polymerase is a JDF-3 DNA polymerase.

24. The kit of claim 23, wherein said JDF-3 DNA polymerase has a sequence of SEQ ID NO:

2 and further comprises one or more amino acid mutations at D141, E143, A485, L408 or P410.

25. The kit of claim 24, wherein said JDF-3 DNA polymerase has one or more amino acid mutations selected from the group consisting of:

D141A or D141T, E143A, L408H or L408F, A485T, and P410L.

26. The kit of claim 25, wherein said JDF-3 DNA polymerase comprises four amino acid mutations at D141A, E 143A, P410L and A485T.

27. The kit of claims 22-26, wherein said Family B DNA polymerase is deficient in 3′

to 5′

exonuclease activity.

28. The kit of claim 22, further comprising at least one chain-terminating nucleotide analog, wherein said chain-terminating nucleotide analog is incorporated into said first primer by said Family B DNA polymerase in a template-dependent manner.

29. The kit of claim 22, wherein at least one chain-terminating nucleotide analog is labeled with a first detectable label.

30. The kit of claim 22, wherein more than one chain-terminating nucleotide analog is labeled, each chain-terminating nucleotide analog being labeled with a different first detectable label.

31. The kit of claim 28, wherein said chain-terminating nucleotide analog is a dideoxynucleotide.

32. The kit of claim 31, wherein said dideoxynucleotide is selected from the group consisting of:

ddATP, ddTTP, ddCTP and ddGTP.

33. The kit of claim 29, wherein said first primer is labeled with a second detectable label.

34. The kit of claim 33, wherein first and second detectable labels generate a signal for identifying said nucleotide at the given position of the template DNA molecule.

35. The kit of claim 22, further comprising a second primer.

36. The kit of claim 35, wherein said first primer is labeled with a second detectable label and said second primer is labeled with a third detectable label, said second and third detectable labels generate a signal for identifying said nucleotide at the given position of the template DNA molecule.

37. The kit of claim 36, wherein said second primer anneals to the immediate 5′

of said nucleotide at the given position of said template DNA molecule.

38. The kit of claim 37, further comprising a DNA ligase.

39. The kit of claim 22, further comprising a reaction buffer for said Family B DNA polymerase.

40. The kit of claim 22, wherein said template DNA molecule is the product of a polymerase chain reaction or a plasmid DNA.

41. The kit of claim 29, 33, or 36, wherein said first or second or third detectable label is one selected from the group consisting of:

a fluorescent label, an isotope, a chemiluminescent label, a quantum dot label, an antigen, or an affinity moiety.

42. The kit of claim 41, wherein said first detectable label is a rhodamine label or a cyanine label.

43. The kit of claim 22, 28 or 39, further comprising a control template and/or at least one control primer.

44. The kit of claim 43, comprising a control template and four control primers.

45. A method of identifying a nucleotide at a given position of a template DNA molecule in a sample, said method comprising:

(a) contacting a first primer with said template DNA molecule, wherein said contacting allows said first primer to anneal to the immediate 3′

of said nucleotide at the given position of said template DNA molecule, so as to form a duplex between said first primer and said template DNA molecule;

(b) incubating said duplex from step (a), in the presence of a Family B DNA polymerase and at least one chain-terminating nucleotide analog, said Family B DNA polymerase having reduced discrimination against non-conventional nucleotides and said terminator is labeled with a first detectable label, wherein said incubating allows the incorporation of a labeled chain-terminating nucleotide analog into said first primer by said DNA polymerase in a template-dependent manner; and

(c) determining the presence or identity of said duplex from step (b) by a signal generated from said first detectable label.

46. The method of claim 45, wherein said Family B DNA polymerase is a JDF-3 DNA polymerase.

47. The method of claim 46, wherein said JDF-3 DNA polymerase has a sequence of SEQ ID NO:

2 and further comprises one or more amino acid mutations at D141, E143, A485, L408 or P410.

48. The method of claim 47, wherein said JDF-3 DNA polymerase has one or more amino acid mutations selected from the group consisting of:

D141A or D141T, E143A, L408H or L408F, A485T, and P410L.

49. The method of claim 48, wherein said JDF-3 DNA polymerase comprises four amino acid mutations at D141A, E 143A, P410L and A485T.

50. The method of claims 45-49, wherein said Family B DNA polymerase is deficient in 3′

to 5′

exonuclease activity.

51. The method of claim 45, wherein at least one chain-terminating nucleotide analog is labeled with a first detectable label.

52. The method of claim 45, wherein more than one chain-terminating nucleotide analog is labeled, each chain-terminating nucleotide analog being labeled with a different first detectable label.

53. The method of claim 45, wherein said chain-terminating nucleotide analog is a dideoxynucleotide.

54. The method of claim 53, wherein said dideoxynucleotide is selected from the group consisting of:

ddATP, ddTTP, ddCTP and ddGTP.

55. The method of claim 45, wherein said first primer is labeled with a second detectable label.

56. The method of claim 55, wherein first and second detectable labels generate a signal for identifying said nucleotide at the given position of the template DNA molecule.

57. The method of claim 45, wherein said template DNA molecule is the product of a polymerase chain reaction or a plasmid.

58. The method of claim 57, further comprising removing PCR primers and dNTPs from the PCR product before step (a).

60. The method of claim 45, 55, or 59, wherein said first or second or third detectable label is one selected from the group consisting of:

a radiolabel, a fluorescent label, a chemiluminescent label, a colorimetric label and an enzymatic label.

61. The method of claim 60, wherein said first detectable label is a rhodamine label or a cyanine label.

59. A method of identifying a nucleotide at a given position of a template DNA molecule in a sample, said method comprising:

(a) contacting a first primer and s second primer with said template DNA molecule, wherein said contacting allows said first primer to anneal to the immediate 3′

of said nucleotide at the given position of said template DNA molecule and said second primer to anneal to the immediate 5′

of said nucleotide at the given position of said template DNA molecule, so as to form a complex between said template DNA molecule and said first and second primers, said first primer being labeled with a second detectable label and said second primer being labeled with a third detectable label.

(b) incubating said complex from step (a), in the presence of a DNA ligase., wherein said incubating allows the ligation between said first and second primers so as to form a single molecule; and

(c) determining the presence or identity of said single molecule from step (b) by a signal generated from said second and third detectable labels.