Polymerase enzymes and reagents for enhanced nucleic acid sequencing

• US 20080108082A1
• Filed: 10/22/2007
• Published: 05/08/2008
• Est. Priority Date: 10/23/2006
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Wegener, Jeff, Peluso, Paul, Bibillo, Arek, Rank, David, Hanzel, David
• Granted Patent: US 8,343,746 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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

Abstract

Compositions that include DNA polymerases having increased residence times for nucleotide analogues, particularly modified recombinant Φ29-type DNA polymerases with such increased residence times, are provided. Methods of making the polymerases and of using the polymerases in sequencing and DNA amplification are also provided. Compositions including α-thiophosphate nucleotide analogues with four or more phosphate groups are described, as are methods for determining the sequence of nucleic acid molecules using such analogues.

Claims

1. A composition comprising:

a modified recombinant Φ

29-type DNA polymerase, which modified recombinant polymerase exhibits a residence time for a nucleotide analogue that is greater than a residence time for the nucleotide analogue exhibited by a corresponding wild-type polymerase.

2. The composition of claim 1, wherein the modified recombinant Φ

29-type DNA polymerase is a modified recombinant Φ

29, B103, GA-1, PZA, Φ

15, BS32, M2Y, Nf, G1, Cp-1, PRD1, PZE, SF5, Cp-5, Cp-7, PR4, PR5, PR722, or L17 polymerase.

3. The composition of claim 1, wherein the residence time of the modified recombinant polymerase for the nucleotide analogue is between about 20 msec and about 300 msec.

4. The composition of claim 3, wherein the residence time of the modified recombinant polymerase for the nucleotide analogue is between about 55 msec and about 100 msec.

5. The composition of claim 1, wherein the residence time of the modified recombinant polymerase for the nucleotide analogue is between about 1.5 and about 4 times the residence time of the corresponding wild-type polymerase for the nucleotide analogue.

6. The composition of claim 1, wherein the modified recombinant polymerase comprises one or more amino acid substitutions at one or more positions selected from the group consisting of:

position 135, position 368, position 372, position 478, position 480, and position 512, wherein numbering of positions is relative to wild-type Φ

29 polymerase.

7. The composition of claim 6, wherein the modified recombinant polymerase comprises one or more amino acid substitutions selected from the group consisting of:

K135D, K135E, T368D, T368E, T372D, T372E, T372R, T372K, K478D, K478E, K478R, L480K, L480R, K512D, and K512E.

8. The composition of claim 1, wherein the modified recombinant polymerase is a modified recombinant Φ

29 polymerase comprising at least one amino acid substitution or combination of substitutions selected from the group consisting of;

K135D, K135E, K512D, K512E, T372D, T372E, L480K, L480R, T368D and L480K, T368E and L480K, T372D and K478R, T372E and K478R, T372R and K478D, T372R and K478E, T372K and K478D, and T372K and K478E.

9. The composition of claim 1, wherein the modified recombinant polymerase comprises at least one mutation relative to the corresponding wild-type polymerase that stabilizes the closed conformation of the modified recombinant polymerase.

10. The composition of claim 1, comprising the nucleotide analogue.

11. The composition of claim 10, wherein the nucleotide analogue comprises a fluorophore.

12. The composition of claim 10, wherein the nucleotide analogue is a phosphate-labeled nucleotide analogue.

13. The composition of claim 10, wherein the nucleotide analogue is a labeled nucleotide analogue having from 3-6 phosphate groups.

14. The composition of claim 1, comprising the nucleotide analogue and a DNA template, wherein the modified recombinant polymerase incorporates the nucleotide analogue into a copy nucleic acid in response to the DNA template.

15. The composition of claim 1, wherein the composition is present in a DNA sequencing system.

16. The composition of claim 15, wherein the sequencing system comprises a zero mode waveguide.

17. A method of sequencing a DNA template, the method comprising:

a) providing a reaction mixture comprising;

the DNA template,

a replication initiating moiety that complexes with or is integral to the template,

a modified recombinant DNA polymerase capable of replicating at least a portion of the template using the moiety in a template-dependent polymerization reaction, wherein the modified recombinant polymerase exhibits a residence time for a first nucleotide analogue that is greater than a residence time for the first nucleotide analogue exhibited by a corresponding wild-type polymerase, and

one or more nucleotides and/or nucleotide analogues, which nucleotides and/or nucleotide analogues comprise the first nucleotide analogue;

b) subjecting the reaction mixture to a polymerization reaction in which the modified recombinant polymerase replicates at least a portion of the template in a template-dependent manner, whereby the one or more nucleotides and/or nucleotide analogues are incorporated into the resulting DNA; and

c) identifying a time sequence of incorporation of the one or more nucleotides and/or nucleotide analogues into the resulting DNA.

18. The method of claim 17, wherein the modified recombinant DNA polymerase exhibits a residence time for a second nucleotide analogue that is greater than a residence time for the second nucleotide analogue exhibited by the corresponding wild-type polymerase, and wherein the one or more nucleotides and/or nucleotide analogues comprise the first and second nucleotide analogues.

19. The method of claim 18, wherein the first and second nucleotide analogues comprise different labels which are distinguished from each other during the identifying step.

20. The method of claim 19, wherein the labels are fluorophores.

21. The method of claim 17, wherein the modified recombinant DNA polymerase is a modified recombinant Taq, DNA Pol I, T7, RB69, or T5 polymerase.

22. The method of claim 17, wherein the modified recombinant DNA polymerase is a modified recombinant Φ

29-type DNA polymerase.

23. The method of claim 22, wherein the modified recombinant Φ

29-type DNA polymerase is a modified recombinant Φ

29, B103, GA-1, PZA, Φ

15, BS32, M2Y, Nf, G1, Cp-1, PRD1, PZE, SF5, Cp-5, Cp-7, PR4, PR5, PR722, or L17 polymerase.

24. The method of claim 22, wherein the modified recombinant polymerase comprises one or more amino acid substitutions at one or more positions selected from the group consisting of:

position 135, position 368, position 372, position 478, position 480, and position 512, wherein numbering of positions is relative to wild-type Φ

29 polymerase.

25. The method of claim 24, wherein the modified recombinant polymerase comprises one or more amino acid substitutions selected from the group consisting of:

K135D, K135E, T368D, T368E, T372D, T372E, T372R, T372K, K478D, K478E, K478R, L480K, L480R, K512D, and K512E.

26. The method of claim 22, wherein the modified recombinant polymerase is a modified recombinant Φ

29 polymerase comprising at least one amino acid substitution or combination of substitutions selected from the group consisting of;

K135D, K135E, K512D, K512E, T372D, T372E, L480K, L480R, T368D and L480K, T368E and L480K, T372D and K478R, T372E and K478R, T372R and K478D, T372R and K478E, T372K and K478D, and T372K and K478E.

27. The method of claim 17, wherein the residence time of the modified recombinant polymerase for the first nucleotide analogue is between about 20 msec and about 300 msec.

28. The method of claim 27, wherein the residence time of the modified recombinant polymerase for the first nucleotide analogue is between about 55 msec and about 100 msec.

29. The method of claim 17, wherein the first nucleotide analogue is a phosphate-labeled nucleotide analogue.

30. The method of claim 17, wherein the first nucleotide analogue is a labeled nucleotide analogue having from 3-6 phosphate groups.

31. The method of claim 17, wherein the subjecting and identifying steps are performed in a zero mode waveguide.

32. A method of making a DNA, the method comprising:

a) providing a reaction mixture comprising;

a template,

a replication initiating moiety that complexes with or is integral to the template,

a modified recombinant Φ

29-type polymerase capable of replicating at least a portion of the template using the moiety in a template-dependent polymerase reaction, wherein the modified recombinant polymerase exhibits a residence time for a first nucleotide analogue that is greater than a residence time for the first nucleotide analogue exhibited by a corresponding wild-type polymerase, and

one or more nucleotides and/or nucleotide analogues, which nucleotides and/or nucleotide analogues comprise the first nucleotide analogue; and

b) reacting the mixture such that the polymerase replicates at least portion of the template in a template-dependent manner, whereby the one or more nucleotides and/or nucleotide analogues are incorporated into the resulting DNA.

33. The method of claim 32, wherein the modified recombinant DNA polymerase exhibits a residence time for a second nucleotide analogue that is greater than a residence time for the second nucleotide analogue exhibited by the corresponding wild-type polymerase, and wherein the one or more nucleotides and/or nucleotide analogues comprises the first and second nucleotide analogues.

34. The method of claim 32, wherein the modified recombinant Φ

29-type DNA polymerase is a modified recombinant Φ

29, B103, GA-1, PZA, Φ

15, BS32, M2Y, Nf, G1, Cp-1, PRD1, PZE, SF5, Cp-5, Cp-7, PR4, PR5, PR722, or L17 polymerase.

35. The method of claim 32, wherein the modified recombinant polymerase is a modified recombinant Φ

29 polymerase comprising at least one amino acid substitution or combination of substitutions selected from the group consisting of;

K135D, K135E, K512D, K512E, T372D, T372E, L480K, L480R, T368D and L480K, T368E and L480K, T372D and K478R, T372E and K478R, T372R and K478D, T372R and K478E, T372K and K478D, and T372K and K478E.

36. The method of claim 32, wherein the residence time of the modified recombinant polymerase for the first nucleotide analogue is between about 20 msec and about 300 msec.

37. The method of claim 36, wherein the residence time of the modified recombinant polymerase for the first nucleotide analogue is between about 55 msec and about 100 msec.

38. The method of claim 32, wherein the first nucleotide analogue is a phosphate-labeled nucleotide analogue.

39. The method of claim 32, wherein the first nucleotide analogue is a labeled nucleotide analogue having from 3-6 phosphate groups.

40. The method of claim 32, wherein the mixture is reacted in a zero mode waveguide.

41. The method of claim 32, the method comprising detecting incorporation of the first nucleotide analogue.

42. A method of making a modified recombinant DNA polymerase, the method comprising:

structurally modeling a first DNA polymerase;

identifying one or more amino acid residue positions in the first DNA polymerase as targets for mutation;

mutating the first polymerase at the one or more positions to introduce at least one intramolecular interaction predicted to stabilize the closed complex of the polymerase or to remove at least one intramolecular interaction predicted to destabilize the closed complex; and

,

determining whether the resulting modified recombinant polymerase displays an increased residence time for a nucleotide analogue as compared to the first polymerase.

43. The method of claim 42, comprising determining the residence time of the modified recombinant polymerase for a natural nucleotide.

44. The method of claim 42, comprising making a library of modified recombinant polymerases, a plurality of members of the library comprising or more mutations at the one or more positions.

45. The method of claim 44, comprising screening the library to identify at least one member exhibiting an increased residence time for the nucleotide analogue.

46. The method of claim 42, wherein the first DNA polymerase is a Φ

29-type DNA polymerase.

47. A method of sequencing a DNA template, the method comprising:

a) providing a reaction mixture comprising;

the DNA template,

a replication initiating moiety that complexes with or is integral to the template,

a DNA polymerase capable of replicating at least a portion of the template using the moiety in a template-dependent polymerization reaction, and

one or more nucleotides and/or nucleotide analogues, which nucleotides and/or nucleotide analogues comprise at least a first α

-thiophosphate nucleotide analogue having at least four phosphate groups;

b) subjecting the reaction mixture to a polymerization reaction in which the polymerase replicates at least a portion of the template in a template-dependent manner, whereby the one or more nucleotides and/or nucleotide analogues are incorporated into the resulting DNA; and

c) identifying a time sequence of incorporation of the one or more nucleotides and/or nucleotide analogues into the resulting DNA.

48. The method of claim 47, wherein the one or more nucleotides and/or nucleotide analogues comprise at least two α

-thiophosphate nucleotide analogues each having at least four phosphate groups.

49. The method of claim 48, wherein the one or more nucleotides and/or nucleotide analogues comprise four α

-thiophosphate nucleotide analogues, each having at least four phosphate groups.

50. The method of claim 48, wherein the at least two nucleotide analogues comprise different labels which are distinguished from each other during the identifying step.

51. The method of claim 50, wherein the labels are fluorophores.

52. The method of claim 47, wherein the DNA polymerase is a Taq, exonuclease-deficient Taq, DNA Pol I, T7, RB69, or T5 polymerase or a Klenow fragment of a DNA Pol I polymerase.

53. The method of claim 47, wherein the DNA polymerase is a Φ

29-type DNA polymerase.

54. The method of claim 47, wherein the first nucleotide analogue is a phosphate-labeled nucleotide analogue.

55. The method of claim 47, wherein the first nucleotide analogue is an α

-thiotetraphosphate analogue.

56. The method of claim 47, wherein the subjecting and identifying steps are performed in a zero mode waveguide.

57. A composition comprising a first α

-thiophosphate nucleotide analogue having at least four phosphate groups.

58. The composition of claim 57, comprising at least two α

-thiophosphate nucleotide analogues each having at least four phosphate groups.

59. The composition of claim 58, comprising four α

-thiophosphate nucleotide analogues, each having at least four phosphate groups.

60. The composition of claim 58, wherein the at least two nucleotide analogues comprise different labels which are distinguishable from each other.

61. The composition of claim 60, wherein the labels are fluorophores.

62. The composition of claim 57, wherein the first nucleotide analogue is a phosphate-labeled nucleotide analogue.

63. The composition of claim 57, wherein the first nucleotide analogue is an α

-thiotetraphosphate analogue.

64. The composition of claim 57, comprising a DNA polymerase, a DNA template and/or a replication initiating moiety that complexes with or is integral to the template.

65. The composition of claim 57, wherein the composition is present in a DNA sequencing system.

66. The composition of claim 65, wherein the sequencing system comprises a zero mode waveguide.

67. A composition as taught in the specification or figures.

68. A method as taught in the specification or figures.