DNA polymerases with increased 3′-mismatch discrimination

• US 10,647,967 B2
• Filed: 01/11/2018
• Issued: 05/12/2020
• 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:
  US15/868,726
• Publication Number:
  US 20180142221A1
• Time in Patent Office: 852 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 method for conducting primer extension, comprising:

contacting a DNA polymerase with a primer, a polynucleotide template, and nucleoside triphosphates under conditions suitable for extension of the primer, thereby producing an extended primer, wherein the DNA polymerase has increased 3′

-mismatch discrimination activity compared with a control DNA polymerase, wherein the DNA polymerase has at least 95% sequence identity to the amino acid sequence of SEQ ID NO;

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

1 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;

1 is V or I.

2. The method of claim 1, wherein the primer extension method is a method for conducting polymerase chain reaction (PCR) isothermal extension, or sequencing reactions.

3. The method of claim 1, wherein the polynucleotide template is RNA or DNA.

4. The method of claim 1, wherein the primer extension method is a method for conducting reverse transcription.

5. The method of claim 1, wherein the nucleoside triphosphates comprise unconventional nucleotides, ribonucleotides and/or labeled nucleotides.

6. The method of claim 1, wherein the primer and/or template comprises one or more nucleotide analogs.

7. The method 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.

8. The method of claim 7, wherein the amino acid corresponding to position 580 of SEQ ID NO:

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

9. The method of claim 8, wherein the amino acid corresponding to position 580 of SEQ ID NO:

I is G.

10. The method of claim 1, wherein the DNA polymerase comprises 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 E;

X₆ is G, S or A;

X₇ is V or I; and

X₈ is L, I or V (SEQ ID NO;

8).

11. The method of claim 1, wherein the DNA polymerase comprises 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 E;

X₆ is G or S;

X₇ is V or I; and

X₈ is L or I (SEQ ID NO;

9).

12. The method of claim 1, wherein the DNA polymerase comprises 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 E (SEQ ID NO;

     10).