Modified polymerases for improved incorporation of nucleotide analogues

• US 20060240439A1
• Filed: 09/10/2004
• Published: 10/26/2006
• Est. Priority Date: 09/11/2003
• Status: Active Grant

• Current Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Original Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Inventors: Earnshaw, David James, Bailey, David Mark Dunstan, Sanches, Raquel Maria, Smith, Geoffrey Paul, Swerdlow, Harold
• Granted Patent: US 8,460,910 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: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Y 207/07007 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase

Abstract

The invention relates to modified polymerase enzymes which exhibit improved incorporation of nucleotide analogues bearing substituents at the 3′ position of the sugar moiety that are larger in size than the naturally occurring 3′ hydroxyl group. Also described are methods of using the polymerases to incorporate nucleotides into polynucleotides, particularly in the context of DNA sequencing.

Claims

1. An altered polymerase enzyme which is capable of improved incorporation of nucleotides which have been modified at the 3′

sugar hydroxyl such that the substituent is larger in size than the naturally occurring 3′

hydroxyl group, compared to a control polymerase enzyme.

2. The polymerase according to claim 1 which exhibits an increased rate of incorporation of nucleotides which have been modified at the 3′

sugar hydroxyl such that the substituent is larger in size than the naturally occurring 3′

hydroxyl group, compared to the control polymerase.

3. The polymerase according to claim 1 which is capable of incorporating nucleotides which have been modified at the 3′

sugar hydroxyl such that the substituent is larger in size than the naturally occurring 3′

hydroxyl group containing each of the four bases A, T, C and G.

4. The polymerase according to claim 1 which is capable of incorporating nucleotides which have been modified at the 3′

sugar hydroxyl such that the substituent is larger in size than the naturally occurring 3′

hydroxyl group at a reaction temperature of 30°

C.

9. The polymerase according to claim 1 which is capable of incorporating nucleotides which have been modified at the 3′

sugar hydroxyl such that the substituent is larger in size than the naturally occurring 3′

hydroxyl group at reaction temperatures across the full range of from 30°

C. to 80°

C.

12. The polymerase according to claim 1 which is a family B type DNA polymerase.

14. The polymerase according to claim 12 wherein the family B type DNA polymerase is an archael DNA polymerase and is selected from Vent, Deep Vent, 9°

N and Pfu polymerase.

19. The polymerase according to claim 1 wherein the control polymerase comprises any one of the following substitution or double substitution mutations functionally equivalent to the Vent™

DNA polymerase amino acid sequence;

Y412V, Y412L, Y412F, Q486E, Q486L, R487K, A488C, A488S, A488L, A488I, A488F, A488V, K490A, K490R, K490N, N494D, S495A, Y496F, Y496L, Y497S, Y497F, Y499L, Y499F, A488C/Y499F or A488L/Y499L.

20. The polymerase according to claim 19 wherein the control polymerase is a Vent™

DNA polymerase including one of the said substitution or double substitution mutations.

21. The polymerase according to claim 1 wherein the control polymerase comprises any one of the following substitution or double substitution mutations functionally equivalent to the 9°

N DNA polymerase amino acid sequence;

A485L or Y409V/A485L

22. The polymerase according to claim 21 where the control polymerase is a 9°

N DNA polymerase comprising the said substitution mutation or double substitution mutation.

25. The polymerase according to claim 1 wherein the control polymerase comprises any one of the following substitution or double substitution mutations functionally equivalent to the Tsp JDF-3 DNA polymerase amino acid sequence:

A485T, P410L or A485T/P410L

27. The polymerase according to claim 1 which comprises one, two or three amino acid substitution mutations in the motif A region, with the proviso that:

if the polymerase is a mutant Vent™

polymerase, the polymerase does not have any of the single amino acid substitutions Y412V, Y412L or Y412F in the absence of any further substitution mutations in the motif A region,

if the polymerase is a mutant 9°

N polymerase, the polymerase does not have the amino acid substitution Y409V in combination with the amino acid substitution A485L in the absence of any further substitution mutations in the motif A region,

if the polymerase is a mutant Tsp JDF-3 polymerase, the polymerase does not have the amino acid substitution P410L, either alone or in combination with the mutation A485T, in the absence of any further substitution mutations in the motif A region.

33. The polymerase according to claim 27 wherein the first amino acid of the motif A region, if mutated, is mutated to an amino acid selected from:

aromatic amino acids, particularly tyrosine (Y) or phenylalanine (F),

amino acids with aliphatic side chains, particularly isoleucine (I), alanine (A) or valine (V), or glutamate (Q),

cysteine (C) or serine (S).

34. The polymerase according to claim 27 wherein the second amino acid of the motif A region, if mutated, is mutated to an amino acid selected from:

alanine (A), serine (S) or glycine(G), most preferably alanine (A).

35. The polymerase according to claim 27 wherein the third amino acid of the motif A region, if mutated, is mutated to an amino acid selected from:

serine (S), alanine (A) or glycine (G),

Amino acids having beta-branched side chains, particularly isoleucine (I), threonine (T), valine (V) or leucine (L).

38. The polymerase according to claim 34 wherein the motif A region has one of the following amino acid sequences:

YST, FAI, AAA, YAS, YAV, YGI, YSG, SGG, CST, IAL, CGG, SAL, SAA, CAA, YAA, QAS, VSS, VAG, VAV, FAV, AGI, YSS, AAT, FSS or VAL.

40. The polymerase according to claim 27 which further comprises at least one amino acid substitution mutation in the motif B region.

46. The polymerase according to claim 1 to which lacks exonuclease activity.

50. The polymerase according to claim 1 wherein the nucleotides which have been modified at the 3′

sugar hydroxyl such that the substituent is larger in size than the naturally occurring 3′

hydroxyl group comprise a modified nucleotide or nucleoside molecule comprising a purine or pyrimidine base and a ribose or deoxyribose sugar moiety having a removable 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—

O—

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 R′

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 3′

OH;

with the proviso that where Z is —

C(R′

)₂—

S—

R″

, both R′

groups are not H.

51. The polymerase according to claim 50 wherein R′

of the modified nucleotide or nucleoside is an alkyl or substituted alkyl.

52. The polymerase according to claim 51 wherein -Z of the modified nucleotide or nucleoside is of formula —

C(R′

)₂—

N₃.

53. The polymerase according to claim 52 wherein Z is an azidomethyl group.

54. The polymerase according to claim 1 wherein the nucleotides which have been modified at the 3′

sugar hydroxyl such that the substituent is larger in size than the naturally occurring 3′

hydroxyl group comprises a nucleotide or nucleoside having a base attached to a detectable label via a cleavable linker, characterised in that the cleavable linker contains a moiety selected from the group comprising;

57. A nucleic acid molecule encoding an altered polymerase as defined in claim 27.

58. A nucleic acid molecule according to claim 57 which encodes an altered 9°

N polymerase, the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO;

13 but having one or more changes in the nucleic acid sequence in the region from nucleotide 1222 to nucleotide 1230, wherein said nucleic acid molecule encodes a mutant 9°

N polymerase enzyme having one, two or three amino acid substitution mutations in the region from amino acid residue 408 to amino acid residue 410, with the proviso that the nucleic acid molecule does not encode a mutant 9°

N polymerase having the amino acid substitution mutations Y409V and A485L in combination.

61. An expression vector comprising the nucleic acid molecule of claim 57.

64. A method for incorporating nucleotides which have been modified at the 3′

sugar hydroxyl such that the substituent is larger in size than the naturally occurring 3′

hydroxyl group into DNA comprising allowing the following components to interact;

A polymerase according to claim 1

DNA template; and

Nucleotide solution containing the nucleotides which have been modified at the 3′

sugar hydroxyl such that the substituent is larger in size than the naturally occurring 3′

hydroxyl group.

65. The method according to claim 64 wherein the incorporation of the nucleotides which have been modified at the 3′

sugar hydroxyl such that the substituent is larger in size than the naturally occurring 3′

hydroxyl group is detected in order to determine the DNA sequence of the DNA template.

5. (canceled)

6. (canceled)

7. (canceled)

8. (canceled)

10. (canceled)

11. (canceled)

13. (canceled)

15. (canceled)

16. (canceled)

17. (canceled)

18. (canceled)

23. (canceled)

24. (canceled)

26. (canceled)

28. (canceled)

29. (canceled)

30. (canceled)

31. (canceled)

32. (canceled)

36. (canceled)

37. (canceled)

39. (canceled)

41. (canceled)

42. (canceled)

43. (canceled)

44. (canceled)

45. (canceled)

47. An altered 9°

N polymerase comprising the amino acid sequence of any one of SEQ ID NO;

20, SEQ ID NO;

22, SEQ ID NO;

18 or SEQ ID NO;

12.

48. An altered 9°

N polymerase comprising the amino acid sequence as set out in SEQ ID NO;

14 but having one or more changes in the amino acid sequence between residues 408 and 410 and optionally between residues 484 and 486, excluding changes which would result in a protein with a valine residue at amino acid 409 and a leucine residue at amino acid 485 in the absence of any other amino acid changes, wherein the altered 9°

N polymerase exhibits improved incorporation of nucleotides which have been modified at the 3′

sugar hydroxyl such that the substituent is larger in size than the naturally occurring 3′

hydroxyl group as compared to a 9°

N polymerase having the amino acid sequence shown as SEQ ID NO;

14.

49. (canceled)

55. (canceled)

56. (canceled)

59. (canceled)

60. A nucleic acid molecule encoding an altered 9°

N polymerase enzyme, the nucleic acid molecule comprising one of nucleotide sequences SEQ ID NO;

19, SEQ ID NO;

21, SEQ ID NO;

17 or SEQ ID NO;

11.

62. (canceled)

63. (canceled)

66-85. -85. (canceled)