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1. WO2020117968 - POLYMERASES, COMPOSITIONS, AND METHODS OF USE

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CLAIMS

1. A recombinant DNA polymerase comprising an amino acid sequence that is at least 80% identical to a 9°N DNA polymerase amino acid sequence SEQ ID NO: l, wherein the DNA polymerase comprises an amino acid substitution mutation at a position functionally equivalent to Ala281, Phe283, Thr349, or Trp397 in the 9°N DNA polymerase amino acid sequence.

2. The polymerase of claim 1, wherein the substitution mutation at the position functionally equivalent to Ala281 comprises a mutation to a non-polar,

hydrophobic, or uncharged amino acid.

3. The polymerase of claim 2, wherein the substitution mutation at the position functionally equivalent to Ala281 comprises a mutation to Gly or Phe.

4. The polymerase of claim 1, wherein the substitution mutation at the position functionally equivalent to Phe283 comprises a mutation to a polar of uncharged amino acid.

5. The polymerase of claim 4, wherein the substitution mutation at the position functionally equivalent to Phe283 comprises a mutation to Ser.

6. The polymerase of claim 1, wherein the substitution mutation at the position functionally equivalent to Thr349 comprises a mutation to a polar or uncharged amino acid.

7. The polymerase of claim 6, wherein the substitution mutation at the position functionally equivalent to Thr349 comprises a mutation to Ser or Asn.

8. The polymerase of claim 1, wherein the substitution mutation at the position functionally equivalent to Thr349 comprises a mutation to a charged amino acid.

9. The polymerase of claim 8, wherein the substitution mutation at the position functionally equivalent to Thr349 comprises a mutation to Lys.

10. The polymerase of claim 1, wherein the substitution mutation at the position functionally equivalent to Trp397 comprises a mutation to a polar or uncharged amino acid.

11. The polymerase of claim 10, wherein the substitution mutation at the position functionally equivalent to Trp397 comprises a mutation to Cys.

12. The polymerase of claim 1, wherein the substitution mutation at the position functionally equivalent to Trp397 comprises a mutation to a non-polar or hydrophobic amino acid.

13. The polymerase of claim 10, wherein the substitution mutation at the position functionally equivalent to Trp397 comprises a mutation to Phe.

14. The polymerase of claim 1, wherein the polymerase comprises at least two, at least three, or four amino acid substitution mutations at positions functionally equivalent to an amino acid selected from A281, F283, Thr349, or Trp397 in the 9°N DNA polymerase amino acid sequence.

15. The polymerase of claim 1, wherein the polymerase comprises the substitution mutation at the position functionally equivalent to Thr349, and further comprises amino acid substitution mutations at positions functionally equivalent to amino acids Metl29, Aspl41, Glul43, Cys223, Leu408, Tyr409, Pro410, Ala485, Tyr497, Arg247, Glu599, and His633 in the 9°N DNA polymerase amino acid sequence.

16. The polymerase of claim 15, wherein the substitution mutation at the position functionally equivalent to Thr349 comprises a mutation to a charged amino acid.

17. The polymerase of claim 16, wherein the substitution mutation at the position functionally equivalent to Thr349 comprises a mutation to Lys.

18. The polymerase of claim 15, wherein the substitution mutation at the position functionally equivalent to Metl29 comprises a mutation to a non-polar or hydrophobic amino acid.

19. The polymerase of claim 18, wherein the substitution mutation at the position functionally equivalent to Metl29 comprises a mutation to Ala.

20. The polymerase of claim 15, wherein the substitution mutation at the position functionally equivalent to Aspl41 comprises a mutation to a non-polar or hydrophobic amino acid.

21. The polymerase of claim 20, wherein the substitution mutation at the position functionally equivalent to Aspl41 comprises a mutation to Ala.

22. The polymerase of claim 15, wherein the substitution mutation at the position functionally equivalent to Glul43 comprises a mutation to a non-polar or hydrophobic amino acid.

23. The polymerase of claim 22, wherein the substitution mutation at the position functionally equivalent to Glul43 comprises a mutation to Ala.

24. The polymerase of claim 15, wherein the substitution mutation at the position functionally equivalent to Cys223 comprises a mutation to a polar or uncharged amino acid.

25. The polymerase of claim 24, wherein the substitution mutation at the position functionally equivalent to Cys223 comprises a mutation to Ser.

26. The polymerase of claim 15, wherein the substitution mutation at the position functionally equivalent to Leu408 comprises a mutation to a non-polar or hydrophobic amino acid.

27. The polymerase of claim 26, wherein the substitution mutation at the position functionally equivalent to Leu408 comprises a mutation to Ala.

28. The polymerase of claim 15, wherein the substitution mutation at the position functionally equivalent to Tyr409 comprises a mutation to a non-polar or hydrophobic amino acid.

29. The polymerase of claim 28, wherein the substitution mutation at the position functionally equivalent to Tyr409 comprises a mutation to Ala.

30. The polymerase of claim 15, wherein the substitution mutation at the position functionally equivalent to Pro410 comprises a mutation to a non-polar or hydrophobic amino acid.

31. The polymerase of claim 30, wherein the substitution mutation at the position functionally equivalent to Pro410 comprises a mutation to lie.

32. The polymerase of claim 15, wherein the substitution mutation at the position functionally equivalent to Ala485 comprises a mutation to a non-polar or hydrophobic amino acid.

33. The polymerase of claim 32, wherein the substitution mutation at the position functionally equivalent to Ala485 comprises a mutation to Val.

34. The polymerase of claim 15, wherein the substitution mutation at the position functionally equivalent to Tyr497 comprises a mutation to a non-polar, hydrophobic, or uncharged amino acid.

35. The polymerase of claim 34, wherein the substitution mutation at the position functionally equivalent to Tyr497 comprises a mutation to Gly.

36. The polymerase of claim 15, wherein the substitution mutation at the position functionally equivalent to Arg247 comprises a mutation to a polar or uncharged amino acid.

37. The polymerase of claim 36, wherein the substitution mutation at the position functionally equivalent to Arg247 comprises a mutation to Tyr.

38. The polymerase of claim 15, wherein the substitution mutation at the position functionally equivalent to Glu599 comprises a mutation to a polar amino acid.

39. The polymerase of claim 38, wherein the substitution mutation at the position functionally equivalent to Glu599 comprises a mutation to Asp.

40. The polymerase of claim 15, wherein the substitution mutation at the position functionally equivalent to His633 comprises a mutation to a non-polar, hydrophobic, or uncharged amino acid.

41. The polymerase of claim 40, wherein the substitution mutation at the position functionally equivalent to His633 comprises a mutation to Gly.

42. The polymerase of any one of claims 1-14, further comprising an amino acid substitution mutation at a position functionally equivalent to Tyr497 and at least one amino acid substitution mutation at a position functionally equivalent to Arg247, Glu599, or His633 in the 9°N DNA polymerase amino acid sequence.

43. The polymerase of claim 42, wherein the substitution mutation at the position functionally equivalent to Tyr497 comprises a mutation to a non-polar, hydrophobic, or uncharged amino acid.

44. The polymerase of claim 43, wherein the substitution mutation at the position functionally equivalent to Tyr497 comprises a mutation to Gly.

45. The polymerase of claim 42, wherein the substitution mutation at the position functionally equivalent to Arg247 comprises a mutation to a polar or uncharged amino acid.

46. The polymerase of claim 45, wherein the substitution mutation at the position functionally equivalent to Arg247 comprises a mutation to Tyr.

47. The polymerase of claim 42, wherein the substitution mutation at the position functionally equivalent to Glu599 comprises a mutation to a polar amino acid.

48. The polymerase of claim 47, wherein the substitution mutation at the position functionally equivalent to Glu599 comprises a mutation to Asp.

49. The polymerase of claim 42, wherein the substitution mutation at the position functionally equivalent to His633 comprises a mutation to a non-polar, hydrophobic, or uncharged amino acid.

50. The polymerase of claim 49, wherein the substitution mutation at the position functionally equivalent to His633 comprises a mutation to Gly.

51. The polymerase of any one of claims 1-14 or 42-50, wherein the polymerase comprises an amino acid substitution mutation at a position functionally equivalent to Tyr497 and at least two or three amino acid substitution mutations at positions functionally equivalent to an amino acid selected from Arg247, Glu599, or His633 in the 9°N DNA polymerase amino acid sequence.

52. The polymerase of any one of claims 1-14 or 42 -51, further comprising at least one amino acid substitution mutation at a position functionally equivalent to Lys620 or Val661 in the 9°N DNA polymerase amino acid sequence.

53. The polymerase of claim 52, wherein the substitution mutation at the position functionally equivalent to Lys620 comprises a mutation to a polar amino acid.

54. The polymerase of claim 53, wherein the substitution mutation at the position functionally equivalent to Lys620 comprises a mutation to Arg.

55. The polymerase of claim 52, wherein the substitution mutation at the position functionally equivalent to Val661 comprises a mutation to a polar amino acid.

56. The polymerase of claim 55, wherein the substitution mutation at the position functionally equivalent to Val661 comprises a mutation to Asp.

57. The polymerase of any one of claims 1-14 or 42-56, wherein the polymerase comprises an amino acid substitution mutation at a position functionally equivalent to Tyr497 and at least two, at least three, at least four, or at least five amino acid substitution mutations at positions functionally equivalent to an amino acid selected from Arg247, Glu599, His633, Lys620, or Val661 in the 9°N DNA polymerase amino acid sequence.

58. The polymerase of any one of claims 1-14 or 42-57, wherein the polymerase further comprises amino acid substitution mutations at positions functionally equivalent to amino acids Metl29, Asp 141, Glul43, Cys223, Leu408, Tyr409, Pro410, or Ala485 in the 9°N DNA polymerase amino acid sequence.

59. A recombinant DNA polymerase comprising an amino acid sequence that is at least 80% identical to a 9°N DNA polymerase amino acid sequence SEQ ID NO:8, wherein the DNA polymerase comprises an amino acid substitution mutation at a position functionally equivalent to Ala281Gly in the 9°N DNA polymerase amino acid sequence.

60. A recombinant DNA polymerase comprising an amino acid sequence that is at least 80% identical to a 9°N DNA polymerase amino acid sequence SEQ ID NO: 8, wherein the DNA polymerase comprises an amino acid substitution mutation at a position functionally equivalent to Ala281Phe in the 9°N DNA polymerase amino acid sequence.

61. A recombinant DNA polymerase comprising an amino acid sequence that is at least 80% identical to a 9°N DNA polymerase amino acid sequence SEQ ID NO: 8, wherein the DNA polymerase comprises an amino acid substitution mutation at a position functionally equivalent to Phe283Ser in the 9°N DNA polymerase amino acid sequence.

62. A recombinant DNA polymerase comprising an amino acid sequence that is at least 80% identical to a 9°N DNA polymerase amino acid sequence SEQ ID NO: 8, wherein the DNA polymerase comprises an amino acid substitution mutation at a position functionally equivalent to Thr349Ser in the 9°N DNA polymerase amino acid sequence.

63. A recombinant DNA polymerase comprising an amino acid sequence that is at least 80% identical to a 9°N DNA polymerase amino acid sequence SEQ ID NO: 8, wherein the DNA polymerase comprises an amino acid substitution mutation at a position functionally equivalent to Thr349Asn in the 9°N DNA polymerase amino acid sequence.

64. A recombinant DNA polymerase comprising an amino acid sequence that is at least 80% identical to a 9°N DNA polymerase amino acid sequence SEQ ID NO: 8, wherein the DNA polymerase comprises an amino acid substitution mutation at a position functionally equivalent to Thr349Lys in the 9°N DNA polymerase amino acid sequence.

65. A recombinant DNA polymerase comprising an amino acid sequence that is at least 80% identical to a 9°N DNA polymerase amino acid sequence SEQ ID NO: 8, wherein the DNA polymerase comprises an amino acid substitution mutation at a position functionally equivalent to Trp397Cys in the 9°N DNA polymerase amino acid sequence.

66. A recombinant DNA polymerase comprising an amino acid sequence that is at least 80% identical to a 9°N DNA polymerase amino acid sequence SEQ ID NO: 8, wherein the DNA polymerase comprises an amino acid substitution mutation at a position functionally equivalent to Trp397Phe in the 9°N DNA polymerase amino acid sequence.

67. A recombinant DNA polymerase comprising an amino acid sequence that is at least 80% identical to a 9°N DNA polymerase amino acid sequence SEQ ID NO: 8, wherein the DNA polymerase comprises an amino acid substitution mutation at a position functionally equivalent to His633Thr in the 9°N DNA polymerase amino acid sequence.

68. A DNA polymerase comprising the amino acid sequence of any one of SEQ ID NOs:9-17.

69. The polymerase of any one of claims 1-68, wherein the polymerase is a family B type DNA polymerase.

70. The polymerase of claim 69, wherein the polymerase is selected from the group consisting of a family B archaeal DNA polymerase, a human DNA polymerase-a, T4 polymerase, RB69 polymerase, and phi29 phage DNA polymerase.

71. The polymerase of claim 70, wherein the family B archaeal DNA polymerase is from a genus selected from the group consisting of Thermococcus, Pyrococcus, and Methanococcus.

72. The polymerase of any of claims 1-71, wherein the polymerase comprises reduced exonuclease activity as compared to a wild type polymerase.

73. A nucleic acid molecule encoding a polymerase as defined in any one of claims 1-72.

74. An expression vector comprising the nucleic acid molecule of claim 73.

75. A host cell comprising the vector of claim 74.

76. A method for incorporating modified nucleotides into DNA comprising allowing the following components to interact: (i) a polymerase according to any one of claims 1-72, (ii) a DNA template; and (iii) a nucleotide solution.

77. The method of claim 76, wherein the DNA template comprises a clustered array.

78. A kit for performing a nucleotide incorporation reaction comprising: a polymerase as defined in any one of claims 1-72 and a nucleotide solution.

79. The kit of claim 78, wherein the nucleotide solution comprises labelled nucleotides.

80. The kit of claim 78, wherein the nucleotides comprise synthetic nucleotides.

81. The kit of claim 78, wherein the nucleotides comprise modified nucleotides.

82. The kit of claim 78, wherein the modified nucleotides have been modified at the 3' sugar hydroxyl such that the substituent is larger in size than the naturally occurring 3' hydroxyl group.

83. The kit of claim 82, wherein the modified nucleotides comprise a modified nucleotide or nucleoside molecule comprising a purine or pyrimidine base and a ribose or deoxyribose sugar moiety having a 7removable 3 '-OH blocking group covalently attached thereto, such that the 3' carbon atom has attached a group of the structure

-O-Z

wherein Z is any of -C(R')2-0-R", -C(R')2-N(R")2, -C(R)2-N(H)R", -C(R')2-S-R" and -C(R)2-F, wherein each R" is or is part of a removable protecting group; each R1 is independently a hydrogen atom, an alkyl, substituted alkyl, arylalkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclic, acyl, cyano, alkoxy, aryloxy, heteroaryloxy or amido group, or a detectable label attached through a linking group; or (R)2 represents an alkylidene group of formula =C(R’“)2 wherein each R’” may be the same or different and is selected from the group comprising hydrogen and halogen atoms and alkyl groups; and

wherein said molecule may be reacted to yield an intermediate in which each R" is exchanged for H or, where Z is -C(R')2-F, the F is exchanged for OH, SH or NH2, preferably OH, which intermediate dissociates under aqueous conditions to afford a molecule with a free 3ΌH;

with the proviso that where Z is -C(R')2-S-R", both R' groups are not H.

84. The kit of claim 83, wherein R of the modified nucleotide or nucleoside is an alkyl or substituted alkyl.

85. The kit of claim 84, wherein -Z of the modified nucleotide or nucleoside is of formula -C(R')2-N3.

86. The kit of claim 85, wherein Z is an azidomethyl group.

87. The kit of claim 81, wherein the modified nucleotides are fluorescently labelled to allow their detection.

88. The kit of claim 81, wherein the modified nucleotides comprise a nucleotide or nucleoside having a base attached to a detectable label via a cleavable linker.

89. The kit of claim 88, wherein the detectable label comprises a fluorescent label.

90. The kit of claim 78, further comprising one or more DNA template molecules and/or primers.