DNA polymerases with increased 3′-mismatch discrimination
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.
1. A thermostable, family A-type DNA polymerase having increased 3′-mismatch discrimination activity compared with a control DNA polymerase, wherein the DNA polymerase comprises a motif in the polymerase domain comprising
G-Y-V-X 1 -T-L-F-G-R-X 4 -X 5 -X 6 -X 7 -P,
wherein:
X 1 is E, R or T;
X 2 is F, Y or L;
X 3 is G or H;
X 4 is R or K;
X 5 is any amino acid other than R;
X 6 is Y or D; and
X 7 is V, I or L (SEQ ID NO:8),
wherein the DNA polymerase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 2, 36, 37, 38, 39, 40 or 41, 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 at position X 5 is R.
2. The DNA polymerase of claim 1 , wherein X 5 is selected from G, A, L, M, W, P, S, T, F, Y, C, N, Q, D, E, K, V, I or H.
3. The DNA polymerase of claim 2 , wherein X 5 is A, C, D, G, or N.
4. The DNA polymerase of claim 1 , further comprising a motif comprising
T-G-R-L-S-S-X 7 -X 8 -P-N-L-Q-N;
wherein
X 7 is S or T; and
X 8 is any amino acid other than D or E (SEQ ID NO:27).
5. The DNA polymerase of claim 4 , wherein the amino acid corresponding to position X 8 is selected from the group consisting of L, G, T, Q, A, S, N, R, and K.
6. The DNA polymerase of claim 5 , wherein the amino acid corresponding to position X 8 is G.
7. The DNA polymerase of claim 1 , wherein the DNA polymerase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 2.
8. The DNA polymerase of claim 7 , wherein the DNA polymerase is a Taq polymerase, and the amino acid at position 578 of SEQ ID NO: 2 is selected from the group consisting of L, G, T, Q, A, S, N, R, and K.
9. The DNA polymerase of claim 8 , wherein the amino acid at position 578 of SEQ ID NO: 2 is G.
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 3 , wherein the amino acid at position 667 of SEQ ID NO: 36 or the amino acid at position 639 of SEQ ID NO: 37 is any amino acid other than N, wherein the amino acid at position 640 of SEQ ID NO: 38 or the amino acid at position 640 of SEQ ID NO: 39 is any amino acid other than D, and wherein the amino acid at position 620 of SEQ ID NO: 40 or the amino acid at position 621 of SEQ ID NO: 41 is any amino acid other than E.
16. The DNA polymerase of claim 15 , wherein the amino acid at position 667 of SEQ ID NO: 36 is G, the amino acid at position 639 of SEQ ID NO: 37 is G, the amino acid at position 640 of SEQ ID NO: 38 is G, the amino acid at position 640 of SEQ ID NO: 39 is G, the amino acid at position 620 of SEQ ID NO: 40 is G, and the amino acid at position 621 of SEQ ID NO: 41 is G.
17. A thermostable, family A-type 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 730 of SEQ ID NO:1 is any amino acid other than R, wherein the DNA polymerase has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 2, 36, 37, 38, 39, 40 or 41, 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 730 of SEQ ID NO:1 is R.
18. The DNA polymerase of claim 17 , wherein the amino acid of the DNA polymerase corresponding to position 730 of SEQ ID NO:1 is selected from G, A, L, M, W, P, S, T, F, Y, C, N, Q, D, E, K, V, I or H.
19. The DNA polymerase of claim 18 , wherein the amino acid of the DNA polymerase corresponding to position 730 of SEQ ID NO:1 is A, C, D, G, or N.
20. The DNA polymerase of claim 19 , wherein the amino acid of the DNA polymerase corresponding to position 580 of SEQ ID NO:1 is G.