DNA POLYMERASES WITH INCREASED 3'-MISMATCH DISCRIMINATION

• US 20120015405A1
• Filed: 06/17/2011
• Published: 01/19/2012
• Est. Priority Date: 06/18/2010
• Status: Active Grant

• Current Assignee: Roche Molecular Systems Inc (Roche Holding AG)
• Original Assignee: Roche Molecular Systems Inc (Roche Holding AG)
• Inventors: REICHERT, FRED, BAUER, KEITH, MYERS, THOMAS W.
• Granted Patent: US 8,735,121 B2
• Time in Patent Office: Days
• US Class Current: 435/91.2
• 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 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Y 207/07007 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase

Abstract

Disclosed are mutant DNA polymerases having increased 3′-mismatch discrimination relative to a corresponding, unmodified polymerase. The mutant polymerases are useful in a variety of disclosed primer extension methods. Also disclosed are related compositions, including recombinant nucleic acids, vectors, and host cells, which are useful, e.g., for production of the mutant DNA polymerases.

Claims

1. A DNA polymerase having increased 3′

-mismatch discrimination activity compared with a control DNA polymerase, wherein the amino acid of the DNA polymerase corresponding to position 667 of SEQ ID NO;

1 is any amino acid other than V or I, and wherein the control DNA polymerase has the same amino acid sequence as the DNA polymerase except that the amino acid of the control DNA polymerase corresponding to position 667 of SEQ ID NO;

1 is V or I.

2. The DNA polymerase of claim 1, wherein the amino acid of the DNA polymerase corresponding to position 667 of SEQ ID NO:

1 is selected from G, A, L, M, F, W, P, S, T, C, Y, N, Q, D, E, K, R, or H.

3. The DNA polymerase of claim 1, comprising a motif in the polymerase domain comprising

X₁—

R-R-X₂-X₃-K-X₄-X₅-N-F-X₆-X₇-X₈-Y-G,

wherein;

X₁ is M or Q;

X₂ is A, V or Q;

X₃ is A or G;

X₄ is T, M or A;

X₅ is any amino acid other than V or I;

X₆ is G, S or A;

X₇ is V or I; and

X₈ is L, I or V (SEQ ID NO;

8).

4. The DNA polymerase of claim 3, wherein X₅ is selected from G, A, L, M, F, W, P, S, T, C, Y, N, Q, D, E, K, R, or H. (SEQ ID NO:

42).

5. The DNA polymerase of claim 1, comprising a motif in the polymerase domain comprising

M-R-R-A-X₃-K-X₄-X₅-N-F-X₆-X₇-X₈-Y-G,

wherein:

X₃ is A or G;

X₄ is T or M;

X₅ is any amino acid other than V or I;

X₆ is G or S;

X₇ is V or I; and

X₈ is L or I (SEQ ID NO;

9).

6. The DNA polymerase of claim 1, comprising a motif in the polymerase domain comprising

M-R-R-A-A-K-T-X₅-N-F-G-V-L-Y-G,

wherein:

X₅ is any amino acid other than V or I (SEQ ID NO;

     10).

7. The DNA polymerase of claim 6, wherein X₅ is E (SEQ ID NO:

11).

8. The DNA polymerase of claim 1, wherein the amino acid corresponding to position 580 of SEQ ID NO:

1 is any amino acid other than D or E.

9. The DNA polymerase of claim 1, wherein the amino acid corresponding to position 580 of SEQ ID NO:

1 is selected from the group consisting of L, G, T, Q, A, S, N, R, and K.

10. The DNA polymerase of claim 6, wherein the DNA polymerase comprises an amino acid sequence at least 80% sequence identical to SEQ ID NO:

1, 2, 3, 4, 5, 6, 7, 36, 37, 38, 39, 40 or 41.

11. The DNA polymerase of claim 6, wherein the polymerase has at least 80% sequence identity to SEQ ID NO:

1.

12. The DNA polymerase of claim 11, wherein the DNA polymerase is a Z05 DNA polymerase, and the amino acid at position 580 is selected from the group consisting of L, G, T, Q, A, S, N, R, and K.

13. A recombinant nucleic acid encoding the DNA polymerase according to claim 1.

14. A method for conducting primer extension, comprising:

contacting a DNA polymerase according to claim 1 with a primer, a polynucleotide template, and nucleoside triphosphates under conditions suitable for extension of the primer, thereby producing an extended primer.

15. The method of claim 14, wherein the primer extension method is a method for conducting polymerase chain reaction (PCR).

16. A kit for producing an extended primer, comprising:

at least one container providing a DNA polymerase according to claim 1.

17. The kit according to claim 16, further comprising one or more additional containers selected from the group consisting of:

(a) a container providing a primer hybridizable, under primer extension conditions, to a predetermined polynucleotide template;

(b) a container providing nucleoside triphosphates; and

(c) a container providing a buffer suitable for primer extension.

18. A reaction mixture comprising a DNA polymerase according to claim 1, at least one primer, a polynucleotide template, and nucleoside triphosphates.