MODIFIED TYPE A DNA POLYMERASES

• US 20110281305A1
• Filed: 11/03/2009
• Published: 11/17/2011
• Est. Priority Date: 11/03/2008
• Status: Active Grant

• Current Assignee: Kapa Biosystems (Roche Holding AG)
• Original Assignee: Kapa Biosystems Co,
• Inventors: Foskett, John, Rush, Gavin, McEwan, Paul, Appel, Maryke, Bourn, William
• Granted Patent: US 10,457,968 B2
• Time in Patent Office: Days
• US Class Current: 435/91.2
• CPC Class Codes: C12N 15/1058 Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms
• CPC Class Codes: C12N 9/1241 Nucleotidyltransferases (2.7.7)
• 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 1/485 involving kinase
• CPC Class Codes: C12Y 207/07007 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: G01N 2333/9126 DNA-directed DNA polymerase (2.7.7.7)

Abstract

The present invention provides improved DNA polymerases, in particular, type A DNA polymerases, that may be better suited for applications in recombinant DNA technologies. Among other things, the present invention provides modified DNA polymerases derived from directed evolution experiments designed to select mutations that confer advantageous phenotypes under conditions used in industrial or research applications.

Claims

1. A modified Taq DNA polymerase showing at least 70% amino acid sequence identity with wild type Taq DNA polymerase (SEQ ID NO:

1) and differing from Taq DNA polymerase in that its amino acid sequence comprises one or more amino acid alterations at one or more positions corresponding to P6, K53, K56, E57, K171, T203, E209, D238, L294, V310, G364, E400 A414, E507, 5515, E742, or E797 of Taq polymerase, relative to the corresponding wild-type enzyme Taq polymeras,

wherein the one or more amino acid alterations increase enzyme activity, processivity, resistance to nucleic acid intercalating dyes, and/or salt-resistance.

3. The modified type A DNA polymerase of claim 1, wherein the one or more positions comprises a position corresponding to E507 of Taq polymerase.

4. The modified type A DNA polymerase of claim 1, wherein the amino acid alterations comprise amino acid substitutions, deletions or insertions.

5. The modified type A DNA polymerase of claim 4, wherein the amino acid alterations are amino acid substitutions.

7. The modified type A DNA polymerase of claim 5, wherein the one or more amino acid substitutions are selected from the group consisting of P6S, K53N, K56Q, E57D, K171R, T2031, E209G, E209K, D238N, L294P, V310A, G364D, G364S, E400K, A414T, E507K, S515G, E742K or E797G, and combinations thereof.

8. The modified type A DNA polymerase of claim 1, wherein the DNA polymerase is modified from a naturally-occurring Taq polymerase.

13. A nucleotide sequence encoding a modified type A DNA polymerase according to claim 1.

14. A vector comprising the nucleotide sequence of claim 13.

15. A cell comprising the nucleotide sequence of claim 14.

16. A cell comprising the vector of claim 14.

24. The modified Taq DNA polymerase of claim 1, wherein the modified Taq DNA polymerase comprises an amino acid sequence selected from the group consisting of SEQ ID NO:

2 (A3E), SEQ ID NO;

3 (G9S), SEQ ID NO;

4 (D5S), SEQ ID NO;

5 (D2), SEQ ID NO;

6 (A5E), SEQ ID NO;

7 (B6S), SEQ ID NO;

8 (E2S), SEQ ID NO;

9 (A3), SEQ ID NO;

10 (H10), SEQ ID NO;

11 (HIS), SEQ ID NO;

12 (F9E), SEQ ID NO;

13 (ASS), SEQ ID NO;

14 (C10E), SEQ ID NO;

15 (F5S), SEQ ID NO;

16 (E7S), SEQ ID NO;

17 (G6S), SEQ ID NO;

18 (E1E), SEQ ID NO;

19 (C7), SEQ ID NO;

20 (E12), SEQ ID NO;

21 (D9), SEQ ID NO;

22 (F10), SEQ ID NO;

23 (H7), SEQ ID NO;

24 (A5) and combinations thereof.

40. The modified type A DNA polymerase of claim 1, wherein the modified type A DNA polymerase comprises an amino acid sequence at least 80% identical to the wild-type Taq DNA polymerase (SEQ ID NO:

1).

41. The modified type A DNA polymerase of claim 1, wherein the modified type A DNA polymerase comprises an amino acid sequence at least 90% identical to the wild-type Taq DNA polymerase (SEQ ID NO:

1).

42. The modified type A DNA polymerase of claim 1, wherein the modified type A DNA polymerase comprises an amino acid sequence at least 95% identical to the wild-type Taq DNA polymerase (SEQ ID NO:

1).

43. The modified type A DNA polymerase of claim 1, wherein the modified type A DNA polymerase comprises an amino acid sequence otherwise identical to the wild-type Taq DNA polymerase (SEQ ID NO:

1).

44. The modified type A DNA polymerase of claim 1, wherein the modified type A DNA comprises E507K amino acid substitution

2. (canceled)

6. (canceled)

9-12. -12. (canceled)

17-23. -23. (canceled)

25-29. -29. (canceled)

30. A method comprising amplifying a DNA fragment using a modified Taq DNA polymerase,

wherein the modified Taq DNA polymerase showing at least 70% amino acid sequence identity with wild type Taq DNA polymerase (SEQ ID NO:

1) and differing from Taq DNA polymerase in that its amino acid sequence comprises one or more amino acid alterations at one or more positions corresponding to P6, K53, K56, E57, K171, T203, E209, D238, L294, V310, G364, E400 A414, E507, 5515, E742, or E797 of Taq polymerase, relative to the corresponding wild-type enzyme.

31. The method of claim 30, wherein the DNA fragment is longer than 5 kb.

32. The method of claim 30, wherein the DNA fragment is longer than 8 kb.

33. The method of claim 30, wherein the DNA fragment is longer than 10 kb.

34. The method of claim 30, wherein the one or more positions comprise a position corresponding to E507 of Taq polymerase.

35. The method of claim 34, wherein the modified type A DNA comprises E507K amino acid substitution.

36. The method of claim 30, wherein the modified type A DNA polymerase comprises an amino acid sequence at least 80% identical to the wild-type Taq DNA polymerase (SEQ ID NO:

1).

37. The method of claim 30, wherein the modified type A DNA polymerase comprises an amino acid sequence at least 90% identical to the wild-type Taq DNA polymerase (SEQ ID NO:

1).

38. The method of claim 30, wherein the modified type A DNA polymerase comprises an amino acid sequence at least 95% identical to the wild-type Taq DNA polymerase (SEQ ID NO:

1).

39. The method of claim 30, wherein the modified type A DNA polymerase comprises an amino acid sequence otherwise identical to the wild-type Taq DNA polymerase (SEQ ID NO:

1).