DNA polymerase with modified processivity

• US 5,972,603 A
• Filed: 05/31/1996
• Issued: 10/26/1999
• Est. Priority Date: 02/09/1996
• Status: Expired due to Fees

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Tabor, Stanley, Richardson, Charles C., Bedford, Ella
• Primary Examiner(s): Marschel, Ardin H.
• Assistant Examiner(s): Riley, Jezia
• Application Number:
  US08/656,555
• Time in Patent Office: 1,243 Days
• Field of Search: 435/5, 435/6, 530/350
• US Class Current: 435/6.11
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase

Abstract

Chimeric DNA polymerase having a DNA polymerase domain and processivity factor binding domain not naturally associated with DNA polymerase domain.

Claims

1. A chimeric DNA polymerase having a DNA polymerase domain and a processivity factor binding domain not naturally associated with said DNA polymerase domain, wherein said processivity factor binding domain binds thioredoxin.

2. A chimeric DNA polymerase having a DNA polymerase domain and a processivity factor binding domain not naturally associated with said DNA polymerase domain, wherein said processivity factor binding domain consists essentially of amino acids 236 to 363 from T7 gene 5 protein.

3. A chimeric DNA polymerase having a DNA polymerase domain and a processivity factor binding domain not naturally associated with said DNA polymerase domain, wherein said polymerase consists essentially of a wild-type DNA polymerase having substituted in a region of the wild-type DNA polymerase corresponding to amino acids 549 to 609 of Klenow fragment of DNA polymerase I, a processivity factor binding domain that corresponds essentially to amino acids 236 to 363 of T7 gene 5 protein.

4. A chimeric DNA polymerase having a DNA polymerase domain and a processivity factor binding domain not naturally associated with said DNA polymerase domain, wherein the processivity factor binding domain consists essentially of amino acid residues 258 to 333 of T7 DNA polymerase.

5. The chimeric DNA polymerase of claim 4 wherein the processivity factor binding domain is inserted in place of residues 571 to 577 of E. coli DNA polymerase I.

6. The chimeric DNA polymerase of claim 4 wherein the processivity factor binding domain is inserted in place of residues 475 to 481 of Taq DNA polymerase.

7. The chimeric DNA polymerase of claim 4 wherein the processivity factor binding domain is inserted in place of residues 477 to 483 of Tth DNA polymerase.

8. A processivity factor modified to increase its heat stability.

9. The processivity factor of claim 8, wherein said factor is thioredoxin and said modification is the replacement of aspartic acid 26 with alanine.

10. The processivity factor of claim 8, wherein said factor is thioredoxin.

11. The processivity factor of claim 10, wherein said factor is thioredoxin and said modification is the replacement of aspartic acid 26 with alanine.

12. A chimeric DNA polymerase having a DNA polymerase domain and a processivity factor binding domain not naturally associated with said DNA polymerase domain, wherein said processivity factor binding domain is inserted in place of residues 571 to 577 of E. coli DNA polymerase I.

13. A chimeric DNA polymerase having a DNA polymerase domain and a processivity factor binding domain not naturally associated with said DNA polymerase domain, wherein said processivity factor binding domain is inserted in place of residues 475 to 481 of Taq DNA polymerase.

14. A chimeric DNA polymerase having a DNA polymerase domain and a processivity factor binding domain not naturally associated with said DNA polymerase domain, wherein said processivity factor binding domain is inserted in place of residues 477 to 483 of Tth DNA polymerase.