Active surface coupled polymerases

• US 20100261247A1
• Filed: 12/21/2006
• Published: 10/14/2010
• Est. Priority Date: 12/22/2005
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Pham, Thang, Peluso, Paul, Otto, Geoffrey, Turner, Stephen, Korlach, Jonas, Rank, David, Hanzel, David
• Granted Patent: US 8,936,926 B2
• Time in Patent Office: Days
• US Class Current: 435/188
• CPC Class Codes: C07K 2319/23 containing a GST-tag
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2565/531 characterised by the capture moiety being a protein for target oligonucleotides

Abstract

Active surface coupled polymerases, surfaces that include such polymerases, and methods of making and using surface-attached polymerases are provided.

Claims

1. An isolated or recombinant DNA polymerase bound to a solid surface, the polymerase comprising:

an active site; and

,

an artificial or recombinant surface coupling domain, wherein the surface coupling domain is distal to the active site and is bound to the solid surface;

wherein the surface coupling domain comprises;

a biotin, a biotinylatable polypeptide sequence, an avidin sequence, a AviTag sequence, a recombinant dimer domain of the protein, a large extraneous polypeptide domain, a GST sequence, a glutathione, an S tag, a recombinant polymerase domain, a dye, an acceptor, a quencher, or a combination thereof and wherein the polymerase is capable of catalyzing a polymerization reaction while bound to the solid surface.

2. The isolated or recombinant DNA polymerase of claim 1, wherein the polymerase comprises a deletion in one or more editing domain of the polymerase, whereby the polymerase displays reduced or eliminated exonuclease activity.

3. The isolated or recombinant DNA polymerase of claim 1, wherein the polymerase comprises exonuclease activity.

4. The isolated or recombinant DNA polymerase of claim 1, wherein the polymerase is homologous to:

a Taq polymerase, an exonuclease deficient Taq polymerase, an E. coli DNA Polymerase 1, a Klenow fragment, a reverse transcriptase, a Φ

29 related polymerase, a wild type Φ

29 polymerase, an exonuclease deficient Φ

29 polymerase, a T7 DNA Polymerase, or a T5 DNA Polymerase.

5. The isolated or recombinant DNA polymerase of claim 1, wherein the DNA polymerase is homologous to a Φ

29 DNA polymerase, and comprises a structural modification relative to the Φ

29 DNA polymerase selected from;

a deletion of residues 505-525, a deletion within residues 505-525, a K135A mutation, an E375H mutation, an E375S mutation, an E375K mutation, an E375R mutation, an E375A mutation, an E375Q mutation, an E375W mutation, an E375Y mutation, an E375F mutation, an L384R mutation, an E486A mutation, an E486D mutation, a K512A mutation, an N62D mutation, a D12A mutation, and combinations thereof.

6. The isolated or recombinant DNA polymerase of claim 1, wherein the artificial surface coupling domain comprises at least one purification tag.

7. The isolated or recombinant DNA polymerase of claim 1, wherein the polymerase comprises two or more surface coupling domains.

8. The isolated or recombinant DNA polymerase of claim 1, wherein the surface coupling domain is at least 5 kDa in size.

9. The isolated or recombinant DNA polymerase of claim 1, wherein the surface coupling domain is at least 10 kDa in size.

10. The isolated or recombinant DNA polymerase of claim 1, wherein the surface coupling domain is at least 50 kDa in size.

11. The isolated or recombinant DNA polymerase of claim 1, wherein the surface coupling domain is at least 100 kDa in size.

13. The isolated or recombinant DNA polymerase of claim 1, wherein the artificial surface coupling domain is located within an N-terminal domain of the polymerase.

14. The isolated or recombinant DNA polymerase of claim 1, wherein the active site is located within a C-terminal domain of the polymerase.

15. The isolated or recombinant DNA polymerase of claim 1, wherein the active site is located within a C-terminal domain of the polymerase, and the artificial surface coupling domain is located within an N-terminal domain of the polymerase.

18. The isolated or recombinant DNA polymerase of claim 1, wherein the polymerase, when bound to the surface, displays a k_cat/K_m that is at least 10% as high as a corresponding DNA polymerase in solution.

19. The isolated or recombinant DNA polymerase of claim 1, wherein a location of the polymerase on the surface is fixed, thereby providing a spatial address of the polymerase on the surface.

20. The isolated or recombinant DNA polymerase of claim 1, wherein the surface comprises a polymer, a ceramic, glass, a bead, a microbead, a polymer bead, a glass bead, a well, a microwell, a slide, a grid, a rotor, a microchannel, or a combination thereof.

21. The isolated or recombinant DNA polymerase of claim 1, wherein the surface is a planar surface.

22. The isolated or recombinant DNA polymerase of claim 1, wherein the surface comprises or is proximal to a zero mode waveguide.

23. The isolated or recombinant DNA polymerase of claim 1, wherein the polymerase, when bound to the surface, displays a k_cat/K_m that is at least 50% as high as a corresponding DNA polymerase in solution.

24. The isolated or recombinant DNA polymerase of claim 1, wherein the polymerase, when bound to the surface, displays a k_cat/K_m that is at least 75% as high as a corresponding DNA polymerase in solution.

25. The isolated or recombinant DNA polymerase of claim 1, wherein the polymerase is coupled to the surface in a fixed orientation relative to the surface.

26. The isolated or recombinant DNA polymerase of claim 1, wherein the fixed orientation results in an active site of the polymerase being distal to the surface.

12. (canceled)

16. (canceled)

17. The isolated or recombinant DNA polymerase of claim 16, wherein binding of the polymerase to a surface by the surface coupling domain orients the polymerase relative to the surface.

27-65. -65. (canceled)