DNA polymerases with increased 3′-mismatch discrimination

• US 9,273,293 B2
• Filed: 04/21/2014
• Issued: 03/01/2016
• 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.
• Primary Examiner(s): HUTSON, RICHARD G
• Application Number:
  US14/257,653
• Publication Number:
  US 20140342409A1
• Time in Patent Office: 680 Days
• Field of Search: None
• US Class Current: 1/1
• 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 recombinant nucleic acid encoding a DNA polymerase having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:

1, wherein the amino acid of the DNA polymerase corresponding to position 667 of SEQ ID NO;

1 is E.

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

1 is any amino acid other than D or E.

3. The recombinant nucleic acid of claim 1, wherein the amino acid of the DNA polymerase 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.

4. The recombinant nucleic acid of claim 3, wherein the amino acid of the DNA polymerase corresponding to position 580 of SEQ ID NO:

1 is G.

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

1.

6. The recombinant nucleic acid of claim 1, wherein the DNA polymerase has increased 3′

-mismatch discrimination activity compared with a control DNA polymerase, 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.

7. The recombinant nucleic acid of claim 3, wherein the amino acid of the DNA polymerase corresponding to position 580 of SEQ ID NO:

1 is L.

8. The recombinant nucleic acid of claim 3, wherein the amino acid of the DNA polymerase corresponding to position 580 of SEQ ID NO:

1 is T.

9. The recombinant nucleic acid of claim 3, wherein the amino acid of the DNA polymerase corresponding to position 580 of SEQ ID NO:

1 is Q.

10. The recombinant nucleic acid of claim 3, wherein the amino acid of the DNA polymerase corresponding to position 580 of SEQ ID NO:

1 is A.

11. The recombinant nucleic acid of claim 3, wherein the amino acid of the DNA polymerase corresponding to position 580 of SEQ ID NO:

1 is S.

12. The recombinant nucleic acid of claim 3, wherein the amino acid of the DNA polymerase corresponding to position 580 of SEQ ID NO:

1 is N.

13. The recombinant nucleic acid of claim 3, wherein the amino acid of the DNA polymerase corresponding to position 580 of SEQ ID NO:

1 is R.

14. The recombinant nucleic acid of claim 3, wherein the amino acid of the DNA polymerase corresponding to position 580 of SEQ ID NO:

1 is K.

15. An expression vector comprising the recombinant nucleic acid of claim 1.

16. A host cell transformed with the expression vector of claim 15.

17. A method for producing a DNA polymerase having increased 3′

-mismatch discrimination activity compared with a control DNA polymerase, the method comprising culturing the host cell of claim 16 under conditions suitable for expression of the recombinant nucleic acid.