Engineered polymerases for improved sequencing
Provided are nucleic acids encoding engineered polymerases comprising at least one modification in a motif A and/or at least one modification in a motif B of the polymerase and engineered polymerases encoded by the nucleic acids. Also provided are engineered DNA polymerases comprising a variant of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, the variant being at least 80% identical to SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3 and comprising an amino acid substitution at one or more positions selected from the group consisting of L408, Y409, P410, R484, A/L485, and I486. Methods, vectors, kits, and compositions comprising the nucleic acids and compositions, methods and kits comprising the engineered polymerases are also provided.
1. A method of identifying a base in a primed template nucleic acid, comprising
(a) forming a ternary complex by contacting a primer-template nucleic acid hybrid with an engineered DNA polymerase and a test nucleotide comprising a fluorescent label, wherein the polymerase comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:1 and comprises amino acid substitutions L408F, Y409F, and P410T or amino acid substitutions L408F, Y409H, and P410V, wherein the polymerase has increased accuracy in pairing nucleotides to template bases as compared to a control polymerase having the amino acid sequence of SEQ ID NO:1, and wherein the test nucleotide is complementary to a next template base;
(b) detecting the ternary complex by detecting the fluorescent label without incorporation of the test nucleotide into the primer, thereby identifying the next template base; and
(c) incorporating into the primer an unlabeled nucleotide that is complementary to the next template base.
2. The method of claim 1 , wherein, in step (b), the primer comprises a blocking group at its 3′ end to preclude incorporation of the test nucleotide.
3. The method of claim 1 , wherein, in step (a), the contacting occurs under conditions that stabilize the ternary complex and that destabilize a binary complex between the primed template nucleic acid and the polymerase.
4. The method of claim 1 , wherein the unlabeled nucleotide comprises a reversible terminator.
5. The method of claim 1 , wherein the ternary complex comprises an inhibitory cation.
6. The method of claim 1 , wherein the ternary complex lacks a catalytic metal ion.
7. The method of claim 1 , wherein the incorporating step further comprises replacing the polymerase of steps (a) and (b) with a different type of polymerase that catalyzes the incorporation of the unlabeled nucleotide into the primer.
8. The method of claim 1 , wherein the polymerase comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:1.
9. The method of claim 1 , wherein the polymerase comprises an amino acid sequence that is at least 97% identical to SEQ ID NO:1.
10. The method of claim 1 , wherein the polymerase comprises amino acid substitutions L408F, Y409F, and P410T.
11. The method of claim 1 , wherein the polymerase comprises amino acid substitutions L408F, Y409H, and P410V.
12. The method of claim 1 , wherein the polymerase comprises an alanine residue at position 141, an alanine residue at position 143, and a leucine residue at position 485.
13. The method of claim 1 , further comprising, after step (c), repeating steps (a)-(c) at least once to identify at least one additional base in the primed template nucleic acid.
14. The method of claim 1 , wherein the polymerase has increased stability as compared to the control polymerase.
15. The method of claim 1 , wherein the polymerase has improved polymerization kinetic rates as compared to the control polymerase.
16. The method of claim 1 , wherein the polymerase has decreased polymerization error rates as compared to the control polymerase.
17. The method of claim 1 , wherein the polymerase has an average error rate of less than 0.75 at 100 cycles of a sequencing by binding process.
18. The method of claim 1 , wherein the polymerase has an average error rate of less than 8 at 150 cycles of a sequencing by binding process.
19. The method of claim 1 , wherein the polymerase has an average error rate of between 1 and 5 at 150 cycles of a sequencing by binding process.