Active surface coupled polymerases

• US 20080199932A1
• Filed: 10/26/2007
• Published: 08/21/2008
• 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 8,323,939 B2
• Time in Patent Office: Days
• US Class Current: 435/176
• 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

1-17. -17. (canceled)

18. A surface comprising an immobilized active DNA polymerase bound thereon, wherein the polymerase displays a k_cat/K_m that is at least 10% as high as a corresponding DNA polymerase in solution.

19. The surface of claim 18, wherein a location of the polymerase on the surface is fixed, thereby providing a spatial address of the polymerase on the surface.

20. The surface of claim 18, 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 surface of claim 18, wherein the surface is a planar surface.

22. The surface of claim 18, wherein the surface comprises or is proximal to a zero mode waveguide.

23. The surface of claim 18, wherein the the polymerase displays a k_cat/K_m that is at least 50% as high as a corresponding DNA polymerase in solution.

24. The surface of claim 18, wherein the the polymerase displays a k_cat/K_m that is at least 75% as high as a corresponding DNA polymerase in solution.

25. The surface of claim 18, wherein the polymerase is coupled to the surface in a fixed orientation relative to the surface.

26. The surface of claim 25, wherein the fixed orientation results in an active site of the polymerase being distal to the surface.

27. The surface of claim 18, wherein the polymerase comprises two or more surface coupling domains that couple the polymerase to the surface.

28. The surface of claim 18, wherein the polymerase comprises a surface coupling domain that is at least 5 kDa in size.

29. The surface of claim 18, wherein the polymerase comprises a surface coupling domain that is at least 10 kDa in size.

30. The surface of claim 18, wherein the polymerase comprises a surface coupling domain that is at least 50 kDa in size.

31. The surface of claim 18, wherein the polymerase comprises a surface coupling domain that is at least 100 kDa in size.

32. The surface of claim 18, wherein the polymerase comprises an active site located within a C-terminal domain of the polymerase, and a surface coupling domain located within an N-terminal domain of the polymerase.

33. The surface of claim 18, wherein the polymerase is homologous to a polymerase selected from:

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, and a T5 DNA Polymerase;

or

wherein the polymerase is homologous to Phi29 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.

34-65. -65. (canceled)