Surface comprising DNA polymerase having mutations bound through an affinity tag

• US 8,323,939 B2
• Filed: 10/26/2007
• Issued: 12/04/2012
• 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): NAFF, DAVID M
• Application Number:
  US11/978,139
• Publication Number:
  US 20080199932A1
• Time in Patent Office: 1,866 Days
• Field of Search: None
• US Class Current: 435/174
• 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. A surface comprising an immobilized active DNA polymerase bound thereon, wherein the polymerase comprises at least 95% sequence identity over a region of at least 150 residues to the wild-type Φ

29 DNA polymerase of SEQ ID NO;

1, wherein the polymerase comprises mutations at sites corresponding to E375 and K512 relative to the wild-type Φ

29 DNA polymerase of SEQ ID NO;

1, wherein the polymerase is bound to the surface through an affinity tag, and wherein the polymerase displays a k_cat/K_m that is at least 25% as high as a corresponding DNA polymerase in solution.

2. The surface of claim 1, wherein a location of the polymerase on the surface provides a spatial address of the polymerase on the surface.

3. The surface 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.

4. The surface of claim 1, wherein the surface is a planar surface.

5. The surface of claim 1, wherein the surface comprises a zero mode waveguide.

6. The surface of claim 1, wherein the polymerase displays a k_cat/K_m that is at least 50% as high as a corresponding DNA polymerase in solution.

7. The surface of claim 1, wherein the polymerase displays a k_cat/K_m that is at least 75% as high as a corresponding DNA polymerase in solution.

8. The surface of claim 1, wherein two or more surface coupling domains couple the polymerase to the surface.

9. The surface of claim 1, wherein the polymerase is coupled to the surface through a surface coupling domain that is at least 5 kDa in size.

10. The surface of claim 1, wherein the polymerase is coupled to the surface through a surface coupling domain that is at least 10 kDa in size.

11. The surface of claim 1, wherein the polymerase is coupled to the surface through a surface coupling domain that is at least 50 kDa in size.

12. The surface of claim 1, wherein the polymerase is coupled to the surface through a surface coupling domain that is at least 100 kDa in size.

13. The surface of claim 1, wherein the affinity tag is located in the N-terminal region of the polymerase.

14. The surface of claim 1, wherein the polymerase comprises a mutation selected from:

a histidine substitution at position E375, a serine substitution at position E375, a lysine substitution at position E375, an arginine substitution at position E375, an alanine substitution at position E375, a glutamine substitution at position E375, a tryptophan substitution at position E375, a tyrosine substitution at position E375, a phenylalanine substitution at position E375, an alanine substitution at position K512, a tyrosine substitution at position K512, a tryptophan substitution at position K512, a phenylalanine substitution at position K512, a leucine substitution at position K512, a histidine substitution at position K512, an aspartate substitution at position K512, a glutamate substitution at position K512, and combinations thereof, wherein amino acid positions are numbered relative to the wild-type Φ

29 DNA polymerase of SEQ ID NO;

1.

15. The surface of claim 1, wherein the polymerase comprises tyrosine residues at sites corresponding to E375 and K512 relative to the wild-type Φ

29 DNA polymerase of SEQ ID NO;

1.