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. An engineered DNA polymerase comprising a variant of SEQ ID NO:1, the variant being at least 95% identical to SEQ ID NO:1, the variant comprising amino acid substitutions L408F, Y409F and P410T wherein the engineered DNA polymerase has increased accuracy in pairing nucleotides to template bases as compared to a control polymerase having amino acid sequence of SEQ ID NO:1.
2. An engineered DNA polymerase comprising a variant of SEQ ID NO:1, the variant being at least 95% identical to SEQ ID NO:1, the variant comprising amino acid amino acid substitutions L408F, Y409H, and P410V, wherein the engineered DNA polymerase has increased accuracy in pairing nucleotides to template bases as compared to a control polymerase having amino acid sequence of SEQ ID NO:1.
3. The engineered polymerase of claim 1 , wherein the variant further comprises modifications at amino acid positions R484 and A/L485, R484 and I486, A/L485 and I486, or R484, A/L485 and I486.
4. The engineered polymerase of claim 1 , wherein the variant further comprises a modification at amino acid position D141 and/or E143.
5. The engineered polymerase of claim 4 , wherein the modifications are D141A and E143A.
6. The engineered polymerase of claim 1 , wherein the engineered DNA polymerase is a B-type family polymerase.
7. The engineered polymerase of claim 1 , wherein the engineered DNA polymerase has increased stability as compared to a control polymerase.
8. The engineered polymerase of claim 1 , wherein the engineered DNA polymerase has improved polymerization kinetic rates as compared to a control polymerase.
9. The engineered polymerase of claim 1 , wherein the engineered DNA polymerase has decreased polymerization error rates as compared to a control polymerase.
10. The engineered polymerase of claim 1 , wherein the engineered DNA polymerase has an average error rate of less than 0.75 at 100 cycles of a sequencing by binding process.
11. The engineered polymerase of claim 1 , wherein the engineered DNA polymerase has an average error rate of less than 8 at 150 cycles of a sequencing by binding process.
12. The engineered polymerase of claim 1 , wherein the engineered DNA polymerase has an average error rate of between 1 and 5 at 150 cycles of a sequencing by binding process.
13. The engineered polymerase of claim 2 , wherein the variant further comprises modifications at amino acid positions R484 and A/L485, R484 and I486, A/L485 and I486, or R484, A/L485 and I486.
14. The engineered polymerase of claim 2 , wherein the variant further comprises a modification at amino acid position D141 and/or E143.
15. The engineered polymerase of claim 2 , wherein the modifications are D141A and E143A.
16. The engineered polymerase of claim 2 , wherein the engineered DNA polymerase is a B-type family polymerase.
17. The engineered polymerase of claim 2 , wherein the engineered DNA polymerase has increased stability as compared to a control polymerase having amino acid sequence of SEQ ID NO:1.
18. The engineered polymerase of claim 2 , wherein the engineered DNA polymerase has improved polymerization kinetic rates as compared to a control polymerase having amino acid sequence of SEQ ID NO:1.
19. The engineered polymerase of claim 2 , wherein the engineered DNA polymerase has decreased polymerization error rates as compared to a control polymerase having amino acid sequence of SEQ ID NO:1.
20. The engineered polymerase of claim 2 , wherein the engineered DNA polymerase has an average error rate of less than 0.75 at 100 cycles of a sequencing by binding process.
21. The engineered polymerase of claim 2 , wherein the engineered DNA polymerase has an average error rate of less than 8 at 150 cycles of a sequencing by binding process.
22. The engineered polymerase of claim 2 , wherein the engineered DNA polymerase has an average error rate of between 1 and 5 at 150 cycles of a sequencing by binding process.