Active surface coupled polymerases

• US 8,936,926 B2
• Filed: 12/21/2006
• Issued: 01/20/2015
• 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: Hanzel, David, Korlach, Jonas, Peluso, Paul, Otto, Geoffrey, Pham, Thang, Rank, David, Turner, Stephen
• Primary Examiner(s): HUTSON, RICHARD G
• Application Number:
  US11/645,125
• Publication Number:
  US 20100261247A1
• Time in Patent Office: 2,952 Days
• Field of Search: None
• US Class Current: 435/194
• 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;

wherein the polymerase comprises an amino acid sequence that is at least 80% identical to SEQ ID NO;

1 and that comprises at least one mutation selected from the group consisting of 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, and a K512A mutation, wherein identification of positions is relative to SEQ ID NO;

1;

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, or a combination thereof;

wherein the surface coupling domain is bound to a binding partner that is coupled or bound to the solid surface, whereby the polymerase is bound to the solid surface;

wherein the polymerase is capable of catalyzing a polymerization reaction while bound to the solid surface; and

wherein the polymerase, when bound to the surface, displays a kcat/Km that is at least 10% as high as a corresponding DNA polymerase in solution;

wherein the surface comprises or is proximal to a zero mode waveguide.

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 comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:

1.

5. The isolated or recombinant DNA polymerase of claim 1, wherein the DNA polymerase comprises an E375Y mutation.

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.

12. 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.

13. The isolated or recombinant DNA polymerase of claim 1, wherein binding of the polymerase to a surface by the surface coupling domain orients the polymerase relative to the surface.

14. 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.

15. The isolated or recombinant DNA polymerase of claim 1, wherein the surface comprises glass, a slide, or a combination thereof.

16. 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.

17. 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.

18. 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.

19. The isolated or recombinant DNA polymerase of claim 18, wherein the fixed orientation results in an active site of the polymerase being distal to the surface.

20. The isolated or recombinant DNA polymerase of claim 1, wherein the surface coupling domain comprises a biotin, a biotinylatable polypeptide sequence, or a combination thereof.