Claims
- 1. A protein consisting of at least two heterologous domains wherein a first domain that is a sequence-non-specific double-stranded nucleic-acid-binding domain is joined to a second domain that is a catalytic nucleic-acid-modifying domain having a processive nature, where the presence of the sequence-non-specific double-stranded nucleic-acid-binding domain enhances the processive nature of the nucleic-acid-modifying domain compared to an identical protein not having a sequence-non-specific nucleic-acid-binding domain joined thereto.
- 2. A protein of claim 1 wherein the nucleic-acid-modifying domain has polymerase activity.
- 3. A protein of claim 1 wherein the nucleic-acid-modifying domain has thermally stable polymerase activity.
- 4. A protein of claim 1 wherein the nucleic-acid-modifying domain comprises a Thermus polymerase domain.
- 5. A protein of claim 1 wherein the nucleic-acid modifying domain comprises a Pyrococcus polymerase domain.
- 6. A protein of claim 1 wherein the nucleic-acid-modifying domain has RNA polymerase, reverse transcriptase, methylase, 3′ or 5′ exonuclease, gyrase, or topoisomerase activity.
- 7. A protein of claim 1 wherein the sequence-non-specific nucleic-acid-binding domain specifically binds to polyclonal antibodies generated against either Sac7d or Sso7d.
- 8. A protein of claim 1 wherein the sequence-non-specific nucleic-acid-binding domain contains a 50 amino acid subsequence containing 50% amino acid similarity to Sso7d.
- 9. A protein of claim 1 wherein the sequence-non-specific nucleic-acid-binding domain specifically binds to polyclonal antibodies generated against Sso7d.
- 10. A protein of claim 1 wherein the sequence-non-specific nucleic-acid-binding domain is Sso7d.
- 11. A protein of claim 10 wherein the the nucleic-acid modifying domain comprises a Pyrococcus polymerase domain
- 12. A protein of claim 1 wherein the sequence-non-specific nucleic-acid-binding domain specifically binds to polyclonal antibodies generated against the PCNA homolog of Pyrococcus furiosus.
- 13. A protein of claim 1 wherein the sequence-non-specific nucleic-acid-binding domain is the PCNA homolog of Pyrococcus furiosus.
- 14. A protein consisting of at least two heterologous domains, wherein a first domain that is a sequence-non-specific double-stranded nucleic-acid-binding domain is joined to a second domain that is a catalytic nucleic-acid-modifying domain, where the presence of the sequence-non-specific nucleic-acid-binding domain stabilizes the double-stranded conformation of a nucleic acid by at least 1° C. compared to an identical protein not having a sequence-non-specific nucleic acid-binding domain joined thereto.
- 15. A protein of claim 14 wherein the nucleic-acid-modifying domain has polymerase activity.
- 16. A protein of claim 14 wherein the nucleic-acid-modifying domain has thermally stable polymerase activity.
- 17. A protein of claim 14 wherein the nucleic-acid-modifying domain has an RNA polymerase, a reverse transcriptase, a methylase, a 3′ or 5′ exonuclease, a gyrase, or a topoisomerase activity.
- 18. A protein of claim 14 wherein the sequence-non-specific nucleic-acid-binding domain specifically binds to polyclonal antibodies generated against either Sac7d or Sso7d.
- 19. A protein of claim 14 wherein the sequence-non-specific nucleic-acid-binding domain contains a 50 amino acid subsequence containing 50% amino acid similarity to Sso7d.
- 20. A protein of claim 14 wherein the sequence-non-specific nucleic-acid-binding domain specifically binds to polyclonal antibodies generated against Sso7d.
- 21. A protein of claim 14 wherein the sequence-non-specific nucleic-acid-binding domain is Sso7d.
- 22. A protein of claim 14 wherein the sequence-non-specific nucleic-acid-binding domain specifically binds to polyclonal antibodies generated against the PCNA homolog of Pyrococcus furiosus.
- 23. A protein of claim 14 wherein the sequence-non-specific nucleic-acid-binding domain is the PCNA homolog of Pyrococcus furiosus.
- 24. A method of modifying a nucleic acid in an aqueous solution by:
(i) contacting the nucleic acid with a protein comprising at least two heterologous domains, wherein a first domain that is a sequence-non-specific nucleic-acid-binding domain is joined to a second domain that is a catalytic nucleic-acid-modifying domain having a processive nature, where the sequence non-specific nucleic-acid-binding domain:
a. binds to double-stranded nucleic acid, and b. enhances the processivity of the enzyme compared to an identical enzyme not having the sequence non-specific nucleic-acid-binding domain fused to it, and
wherein the solution is at a temperature and of a composition that permits the binding domain to bind to the nucleic acid and the enzyme to function in a catalytic manner, and (ii) permitting the catalytic domain to modify the nucleic acid in the solution.
- 25. A method of claim 24 wherein the nucleic-acid-modifying domain has polymerase activity.
- 26. A method of claim 24 wherein the nucleic-acid-modifying domain has thermally stable polymerase activity.
- 27. A protein of claim 24 wherein the nucleic-acid-modifying domain comprises a Thermus polymerase domain.
- 28. A protein of claim 24 wherein the nucleic-acid-modifying domain comprises a Pyrococcus polymerase domain.
- 29. A method of claim 24 wherein the nucleic-acid-modifying enzyme has an RNA polymerase, a reverse transcriptase, a methylase, an exonuclease, a gyrase, a topoisomerase, or other processive enzyme activity that interacts with nucleic acid.
- 30. A method of claim 24 wherein the sequence-non-specific nucleic-acid-binding domain specifically binds to polyclonal antibodies generated against either Sac7d or Sso7d.
- 31. A method of claim 24 wherein the sequence-non-specific nucleic-acid-binding domain contains a 50 amino acid subsequence containing 50% amino acid similarity to Sso7D.
- 32. A method of claim 24 wherein the sequence-non-specific nucleic-acid-binding domain specifically binds to polyclonal antibodies generated against Sso7d.
- 33. A method of claim 24 wherein the sequence-non-specific nucleic-acid-binding domain is Sso7d.
- 34. A method of modifying a nucleic acid by:
(i) contacting the nucleic acid with an aqueous solution containing a protein having at least two heterologous domains, wherein a first domain that is a sequence-non-specific double-stranded nucleic acid-binding domain is joined to a second domain that is a catalytic nucleic-acid-modifying domain, where the presence of the sequence-non-specific nucleic-acid-binding domain s stabilizes the double-stranded conformation of a nucleic acid by at least 1° C. compared to an otherwise identical protein not having the sequence-non-specific nucleic-acid-binding domain joined to it; and, wherein the solution is at a temperature and of a composition that permits the binding domain to bind to the nucleic acid and the enzyme to function in a catalytic manner; and, (ii) permitting the catalytic domain to modify the nucleic acid in the solution.
- 35. A method of claim 34 wherein the nucleic-acid-modifying domain has polymerase activity.
- 36. A method of claim 34 wherein the nucleic-acid-modifying domain has thermally stable polymerase activity.
- 37. A protein of claim 34 wherein the nucleic-acid-modifying domain comprises a Thermus polymerase domain.
- 38. A method of claim 34 wherein the nucleic-acid-modifying enzyme has an RNA polymerase, a reverse transcriptase, a methylase, a exonuclease, a gyrase, or a topoisomerase activity.
- 39. A method of claim 34 wherein the sequence-non-specific nucleic-acid-binding domain specifically binds to polyclonal antibodies generated against either Sac7d or Sso7d.
- 40. A method of claim 34 wherein the sequence-non-specific nucleic-acid-binding domain contains a 50 amino acid subsequence containing 50% amino acid similarity to Sso7D.
- 41. A method of claim 34 wherein the sequence-non-specific nucleic-acid-binding domain contains a 50 amino acid subsequence containing 75% amino acid similarity to Sso7D.
- 42. A method of claim 34 wherein the sequence-non-specific nucleic-acid-binding domain specifically binds to polyclonal antibodies generated against Sso7d.
- 43. A method of claim 34 wherein the sequence-non-specific nucleic-acid-binding domain is Sso7d.
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional Application Serial No. 60/207,567 filed May 26, 2000, the disclosure of which is herein incorporated by reference.
Provisional Applications (1)
|
Number |
Date |
Country |
|
60207567 |
May 2000 |
US |
Continuations (1)
|
Number |
Date |
Country |
Parent |
09640958 |
Aug 2000 |
US |
Child |
10256705 |
Sep 2002 |
US |