DNA polymerases with increased 3′-mismatch discrimination

• US 9,902,944 B2
• Filed: 02/09/2016
• Issued: 02/27/2018
• Est. Priority Date: 06/18/2010
• Status: Active Grant

• Current Assignee: Roche Molecular Systems (Roche Holding AG)
• Original Assignee: Roche Molecular Systems (Roche Holding AG)
• Inventors: Reichert, Fred, Bauer, Keith, Myers, Thomas W.
• Primary Examiner(s): Hutson, Richard G
• Application Number:
  US15/019,329
• Publication Number:
  US 20160160190A1
• Time in Patent Office: 749 Days
• Field of Search: None
• 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 DNA polymerase has at least 90% sequence identity to the amino acid sequence of SEQ ID NO;

6 and the amino acid of the DNA polymerase corresponding to position 667 of SEQ ID NO;

6 is E, 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;

6 is V.

2. 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 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).

3. The DNA polymerase of claim 2, 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).

4. 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).

5. 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).

6. The DNA polymerase of claim 5, wherein X₅ is E (SEQ ID NO:

11).

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

6 is any amino acid other than D or E.

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

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

9. The DNA polymerase of claim 5, wherein the polymerase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:

6.

10. 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.

11. The method of claim 10, wherein the primer extension method is a method for conducting polymerase chain reaction (PCR).

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

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

13. The kit according to claim 12, 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.

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

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

6 is G.

16. The DNA polymerase of claim 1, wherein the polymerase has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:

6.

17. The DNA polymerase of claim 1, wherein the polymerase has at least 97% sequence identity to the amino acid sequence of SEQ ID NO:

6.