DNA binding protein-polymerase chimeras
View Patent ↗The invention relates to compositions and methods directed to chimeric DNA polymerases, which comprise a mutated DNA binding polypeptide domain and a mutated or wild-type DNA polymerase polypeptide domain.
1. A chimeric DNA polymerase comprising a DNA binding domain and a polymerase domain, wherein said DNA binding domain has the wild-type sequence of Sso7d (SEQ ID NO:2) or of a wild-type Sso7d-like protein selected from the group consisting of Ssh7b (SEQ ID NO:8), RiboP3 (SEQ ID NO:9), Stole (SEQ ID NO:10), Sac7d (SEQ ID NO:12), Sac7e (SEQ ID NO:14), and Sac7a (SEQ ID NO:16), with the exception of seven, eight, or nine substitution mutations at positions corresponding to seven, eight, or all nine of the following amino acid positions: 13, 16, 40, 41, 45, 55, 56, 61, and 63 of SEQ ID NO:2.
2. The chimeric DNA polymerase of claim 1 , wherein said substitution mutations are selected from the group consisting of:
an E or Q the position corresponding to position 13 of SEQ ID NO:2;
an I or T at the position corresponding to position 16 of SEQ ID NO:2;
a T or S at the position corresponding to position 40 of SEQ ID NO:2;
a G or A at the position corresponding to position 41 of SEQ ID NO:2;
a V or L at the position corresponding to position 45 of SEQ ID NO:2;
an L or V at the position corresponding to position 55 of SEQ ID NO:2;
a Q or D at the position corresponding to position 56 of SEQ ID NO:2;
a Q or E at the position corresponding to position 61 of SEQ ID NO:2; and
a K or R at the position corresponding to position 63 of SEQ ID NO:2.
3. A chimeric DNA polymerase comprising a DNA binding domain and a polymerase domain, wherein said DNA binding domain has the sequence of Sso7d (SEQ ID NO:2) or of a wild-type Sso7d-like protein selected from the group consisting of Ssh7b (SEQ ID NO:8), RiboP3 (SEQ ID NO:9), Stole (SEQ ID NO:10), Sac7d (SEQ ID NO:12), Sac7e (SEQ ID NO:14), and Sac7a (SEQ ID NO:16), with the exception of seven substitution mutations at the positions corresponding to amino acid positions 13, 16, 40, 45, 55, 56, and 63 of SEQ ID NO:2.
4. The chimeric DNA polymerase of claim 3 , wherein said substitution mutations are selected from the group consisting of:
an E or Q the position corresponding to position 13 of SEQ ID NO:2;
an I or T at the position corresponding to position 16 of SEQ ID NO:2;
a T or S at the position corresponding to position 40 of SEQ ID NO:2;
a V or L at the position corresponding to position 45 of SEQ ID NO:2;
an L or V at the position corresponding to position 55 of SEQ ID NO:2;
a Q or D at the position corresponding to position 56 of SEQ ID NO:2;
a K or R at the position corresponding to position 63 of SEQ ID NO:2.
5. A chimeric DNA polymerase comprising a DNA binding domain and a polymerase domain, wherein said DNA binding domain comprises the amino acid sequence of SEQ ID NO:4.
6. A chimeric DNA polymerase comprising a DNA binding domain and a polymerase domain, wherein said DNA binding domain has the sequence of Sso7d (SEQ ID NO:2) or of a wild-type Sso7d-like protein selected from the group consisting of Ssh7b (SEQ ID NO:8), RiboP3 (SEQ ID NO:9), Stole (SEQ ID NO:10), Sac7d (SEQ ID NO:12), Sac7e (SEQ ID NO:14), and Sac7a (SEQ ID NO:16), with the exception of nine substitution mutations at the positions corresponding to amino acid positions 13, 16, 40, 41, 45, 55, 56, 61 and 63 of SEQ ID NO:2.
7. The chimeric DNA polymerase of claim 1 , wherein said polymerase domain comprises an archaeal DNA polymerase.
8. The chimeric DNA polymerase of claim 1 , wherein said polymerase domain has thermally stable polymerase activity.
9. The chimeric DNA polymerase of claim 1 , wherein said polymerase domain comprises a Pfu DNA polymerase.
10. The chimeric DNA polymerase of claim 9 , wherein said polymerase domain is at least 95% identical with the amino acid sequence of SEQ ID NO:18.
11. The chimeric DNA polymerase of claim 1 , wherein said polymerase domain comprises a Taq DNA polymerase.
12. A chimeric DNA polymerase comprising the amino acid sequence of SEQ ID NO:20.
13. A composition comprising the DNA polymerase of claim 1 , or 12 .
14. A kit comprising the chimeric DNA polymerase of claim 1 , or 12 , and packaging materials therefor.
15. A method for DNA synthesis comprising:
a) providing a chimeric DNA polymerase according to claim 1 , or 12 ; and
b) contacting said chimeric DNA polymerase with a nucleic acid template, wherein said enzyme permits DNA synthesis.
16. The chimeric DNA polymerase of claim 1 , wherein said polymerase domain comprises a mutant polymerase.
17. The chimeric DNA polymerase of claim 16 , wherein said mutant polymerase has V93R, V93E, V93D, V93K or V93N mutations resulting in reduced uracil detection activity.
18. The chimeric DNA polymerase of claim 16 , wherein said mutant polymerase has D141A and/or E143A mutations resulting in reduced 3′-5′ exonuclease activity.
19. The chimeric DNA polymerase of claim 6 , wherein said chimeric DNA polymerase has
an E or Q the position corresponding to position 13 of SEQ ID NO:2;
an I or T at the position corresponding to position 16 of SEQ ID NO:2;
a T or S at the position corresponding to position 40 of SEQ ID NO:2;
a G or A at the position corresponding to position 41 of SEQ ID NO:2;
a V or L at the position corresponding to position 45 of SEQ ID NO:2;
an L or V at the position corresponding to position 55 of SEQ ID NO:2;
a Q or D at the position corresponding to position 56 of SEQ ID NO:2;
a Q or E at the position corresponding to position 61 of SEQ ID NO:2; and
a K or R at the position corresponding to position 63 of SEQ ID NO:2.