Claims
- 1. A hybrid polymerase having polymerase activity, wherein the polymerase comprises SEQ ID NO:23 and is at least 80% identical over 700 contiguous amino acids of the Pyrococcus furiosus (Pfu) polymerase sequence set forth in SEQ ID NO: 24 or at least 80% identical over 700 contiguous amino acids of the Pyrococcus sp. GB-D polymerase sequence set forth in SEQ ID NO:25, with the proviso that
(a) when the polymerase is at least 85% identical to SEQ ID NO:24, the sequence comprises at least one hybrid position that is mutated from the native Pfu residue to the residue that occurs at the corresponding position of SEQ ID NO:25, wherein the hybrid position is one of the residues designated as “X” in SEQ ID NO:26; or (b) when the polymerase is at least 85% identical to SEQ ID NO:25, the sequence comprises at least one hybrid position that is mutated from the native Pyrococcus sp. GB-D residue to the residue that occurs at the corresponding position of SEQ ID NO:24, wherein the hybrid position is one of the residues designated as “X” in SEQ ID NO:26.
- 2. The hybrid polymerase of claim 1, wherein the polymerase is at least 90% identical over 700 contiguous amino acids of the Pfu sequence set forth in SEQ ID NO:24 or at least 90% identical over 700 contiguous amino acids of the Pyrococcus sp. GB-D sequence set forth in SEQ ID NO:25.
- 3. The hybrid polymerase of claim 1, wherein the hybrid polymerase comprises at least ten hybrid positions that are mutated from the native reside of SEQ ID NO:24 or SEQ ID NO:25 to the corresponding residue of SEQ ID NO:25 or SEQ ID NO:24.
- 4. The hybrid polymerase of claim 1, wherein the hybrid polymerase comprises at least twenty hybrid positions that are mutated from the native reside of SEQ ID NO:24 or SEQ ID NO:25 to the corresponding residue of SEQ ID NO:25 or SEQ ID NO:24.
- 5. The hybrid polymerase of claim 1, wherein the hybrid polymerase comprises at least forty hybrid positions that are mutated from the native reside of SEQ ID NO:24 or SEQ ID NO:25 to the corresponding residue of SEQ ID NO:25 or SEQ ID NO:24.
- 6. The hybrid polymerase of claim 1, wherein the hybrid polymerase comprises at least fifty hybrid positions that are mutated from the native reside of SEQ ID NO:24 or SEQ ID NO:25 to the corresponding residue of SEQ ID NO:25 or SEQ ID NO:24.
- 7. The hybrid polymerase of claim 1, wherein the hybrid polymerase comprises an amino acid sequence of SEQ ID NO:2, SEQ ID NO:12, SEQ ID NO:16, or SEQ ID NO:18; or the polymerase region of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:14, or SEQ ID NO:20.
- 8. The hybrid polymerase of claim 1, further comprising a DNA binding domain.
- 9. The hybrid polymerase of claim 8, wherein the DNA binding domain is selected from the group consisting of Sso7d, Sac7d, and Sac7e.
- 10. The hybrid polymerase of claim 9, wherein the DNA binding domain is Sso7d.
- 11. The hybrid polymerase of claim 10 comprising an amino acid sequence of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:14, or SEQ ID NO:20.
- 12. An isolated nucleic acid enclosing a hybrid polymerase as set forth in claim 1 or claim 8.
- 13. An expression vector comprising the nucleic acid of claim 12.
- 14. A host cell transfected with the vector of claim 13.
- 15. An isolated nucleic acid encoding a polypeptide comprising an amino acid sequence at least 94% identical to SEQ ID NO:2, wherein the polypeptide exhibits polymerase activity.
- 16. The isolated nucleic acid of claim 15, wherein the polypeptide comprises SEQ ID NO:2.
- 17. The isolated nucleic acid of claim 15, wherein the nucleic acid comprises SEQ ID NO:1.
- 18. The isolated nucleic acid of claim 15, wherein the polypeptide further comprises a DNA binding domain.
- 19. The isolated nucleic acid of claim 18, wherein the DNA binding domain is selected from the group consisting of Sso7d, Sac7d, and Sac7e.
- 20. The isolated nucleic acid of claim 19, wherein the DNA binding domain is Sso7d.
- 21. The isolated nucleic acid of claim 19, wherein the nucleic acid comprises SEQ ID NO:3.
- 22. The isolated nucleic acid of claim 19, wherein the nucleic acid encodes a polypeptide comprising SEQ ID NO:4.
- 23. An expression vector comprising the nucleic acid of claim 15.
- 24. A host cell transfected with the vector of claim 23.
- 25. An isolated polypeptide, wherein the polypeptide comprises an amino acid sequence at least 94% identical to SEQ ID NO:2, and wherein the polypeptide has polymerase activity.
- 26. The isolated polypeptide of claim 25, wherein the polypeptide comprises SEQ ID NO:2.
- 27. The isolated polypeptide of claim 25, further comprising a DNA binding domain.
- 28. The isolated polypeptide of claim 27, wherein the DNA binding domain is selected from the group consisting of Sso7d, Sac7d, or Sac7e.
- 29. The isolated polypeptide of claim 27, wherein the DNA binding domain is fused to the carboxy-terminus of the polypeptide.
- 30. The isolated polypeptide of claim 29, wherein the DNA binding domain is Sso7d.
- 31. The isolated polypeptide of claim 30, wherein the polypeptide comprises SEQ ID NO:4.
- 32. A method of amplifying a target sequence using a hybrid polymerase, the method comprising the steps of:
providing a polymerase according to claim 1 or claim 25, combining the polymerase in an amplification reaction mixture, and amplifying the target sequence.
Parent Case Info
[0001] CROSS-REFERENCES TO RELATED APPLICATIONS
[0002] This application claims the benefit of U.S. Provisional Application No. 60/398,687, filed Jul. 25, 2002; and U.S. Provisional Application 60/483,287, filed Jun. 27, 2003, each of which applications is herein incorporated by reference.
Provisional Applications (2)
|
Number |
Date |
Country |
|
60483287 |
Jun 2003 |
US |
|
60398687 |
Jul 2002 |
US |