Polymerases for nucleotide analogue incorporation

• US 8,921,086 B2
• Filed: 12/21/2006
• Issued: 12/30/2014
• 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 K., Otto, Geoff, Peluso, Paul, Pham, Thang, Rank, David R., Mitsis, Paul, Christians, Fred, Bibillo, Arek, Park, Insil, Clark, Sonya, Lyle, John
• Primary Examiner(s): HUTSON, RICHARD G
• Application Number:
  US11/645,223
• Publication Number:
  US 20070196846A1
• Time in Patent Office: 2,931 Days
• Field of Search: None
• US Class Current: 435/194
• CPC Class Codes: C07K 2319/20 containing a tag with affinity for a non-protein ligand
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Y 207/07007 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: Y02P 20/52 using catalysts, e.g. selective catalysts

Abstract

Compositions that include polymerases with features for improving entry of nucleotide analogues into active site regions and for coordinating with the nucleotide analogues in the active site region are provided. Methods of making the polymerases and of using the polymerases in sequencing and DNA replication and amplification as well as kinetic models of polymerase activity and computer-implemented methods of using the models are also provided.

Claims

1. A composition comprising a recombinant DNA polymerase, which recombinant polymerase comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:

1 and comprises an amino acid substitution at position 375 and an amino acid substitution at position 512, wherein numbering of positions is relative to SEQ ID NO;

1, wherein the recombinant polymerase exhibits polymerase activity; and

a nucleotide analogue that is labeled on a phosphate group, wherein the base moiety of the analogue is selected from the group consisting of adenine, thymine, guanine, and cytidine.

2. The composition of claim 1, wherein the amino acid substitution at position 375 is 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, and an E375F mutation.

3. The composition of claim 2, wherein the recombinant DNA polymerase further comprises an L384R mutation.

4. The composition of claim 1, wherein the recombinant DNA polymerase comprises a structural modification that reduces exonuclease activity of the recombinant polymerase relative to the wild-type polymerase of SEQ ID NO:

1.

5. The composition of claim 1, wherein the polymerase comprises an N62 mutation relative to the wild-type Φ

29 DNA polymerase of SEQ ID NO;

1.

6. The composition of claim 1, wherein the recombinant DNA polymerase comprises one or more exogenous affinity tag sequence.

7. The composition of claim 6, wherein the affinity tag sequence is selected from:

a 6 His tag sequence, a GST tag, an HA tag sequence, a plurality of 6 His tag sequences, a plurality of GST tags, a plurality of HA tag sequences and combinations thereof.

8. The composition of claim 1, wherein the nucleotide analogue comprises a fluorophore or a dye moiety.

9. The composition of claim 1, wherein the recombinant polymerase displays a modified property for the nucleotide analogue as compared to the wild-type polymerase of SEQ ID NO:

1, wherein the modified property is selected from;

K_m, k_cat, V_max, recombinant polymerase processivity in the presence of the nucleotide analogue, average template read-length by the recombinant polymerase in the presence of the nucleotide analogue, specificity of the recombinant polymerase for the nucleotide analogue, rate of binding of the nucleotide analogue, rate of product release, and branching rate.

10. The composition of claim 9, wherein the modified property comprises a reduced K_m for the nucleotide analogue.

11. The composition of claim 9, wherein the modified property comprises an increased k_cat/K_m or V_max/K_m for the nucleotide analogue.

12. The composition of claim 1, comprising a DNA template, wherein the recombinant polymerase incorporates the nucleotide analogue into a copy nucleic acid in response to the template DNA.

13. The composition of claim 12, wherein the template is a circular template.

14. The composition of claim 1, wherein the composition is present in a DNA sequencing system.

15. The composition of claim 14, wherein the sequencing system comprises a Zero Mode Waveguide.

16. The composition of claim 1, wherein the recombinant DNA polymerase comprises an E375Y mutation and a K512Y mutation, relative to the wild-type Φ

29 DNA polymerase of SEQ ID NO;

1.

17. The composition of claim 16, wherein the recombinant DNA polymerase additionally comprises a D12A or N62D mutation relative to the wild-type Φ

29 DNA polymerase of SEQ ID NO;

1.

18. The composition of claim 1, wherein the nucleotide analogue is a tetraphosphate labeled analogue comprising a label on a delta phosphate of the analogue.

19. The composition of claim 18, wherein the tetraphosphate analogue is A488dC4P, A568dC4P, A633dC4P, A546dG4P, or A594dT4P.

20. The composition of claim 1, wherein the amino acid substitution at position 512 is selected from the group consisting of:

a K512A mutation, a K512Y mutation, a K512W mutation, a K512F mutation, a K512L mutation, a K512H mutation, a K512D mutation, and a K512E mutation.

21. The composition of claim 1, wherein the recombinant polymerase comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:

1.