Enzymes resistant to photodamage

• US 9,127,259 B2
• Filed: 03/30/2009
• Issued: 09/08/2015
• Est. Priority Date: 03/31/2008
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Bjornson, Keith, Clark, Sonya, Park, Insil, Christians, Fred
• Primary Examiner(s): HUTSON, RICHARD G
• Application Number:
  US12/384,110
• Publication Number:
  US 20100093555A1
• Time in Patent Office: 2,353 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12N 9/1276 RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2535/113 Cycle sequencing

Abstract

Provided are compositions comprising modified DNA polymerases that exhibit improved photostability compared to the parental polymerases from which they were derived. Provided are methods for generating enzymes, such as DNA polymerases, with the aforementioned phenotype. Provided are methods of using polymerases with increased resistance to photodamage to make a DNA or to sequence a DNA template.

Claims

1. A composition comprising a modified recombinant DNA polymerase, which modified recombinant polymerase exhibits polymerase activity, which modified recombinant polymerase comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:

1 or SEQ ID NO;

21, and which modified recombinant polymerase comprises at least one amino acid substitution relative to a parental polymerase, wherein the substitution is selected from the group consisting of;

M246L, F248L, W367S, Y369V, Y482V, W483S, W483F, W483L, W483V, W483I, W483P, W483Q, H485G, H485N, H485K, H485R, H485A, H485E, H485S, H485I, H485P, H485Q, H485T, H485F, H485L, Y505V, M506L, Y521V, and F526L, wherein numbering of positions is relative to SEQ ID NO;

1.

2. The composition of claim 1, wherein the modified recombinant polymerase is a modified recombinant Φ

29 polymerase comprising at least one amino acid substitution or combination of substitutions selected from the group consisting of;

M246L, F248L, W367S, Y369V, Y482V, W483S, W483F, W483L, W483V, W483I, W483P, W483Q, H485G, H485N, H485K, H485R, H485A, H485E, H485S, H485I, H485P, H485Q, H485T, H485F, H485L, Y505V, M506L, Y521V, F526L, M246L and F248L, W367S and Y369V, Y482V and W483S, Y482V and H485G, W483S and H485G, Y505V and M506L, and Y521V and F526L, wherein numbering of positions is relative to SEQ ID NO;

1.

3. The composition of claim 1, wherein the parental polymerase is a wild type or an exonuclease deficient Φ

29 polymerase.

4. The composition of claim 1, comprising a phosphate-labeled nucleotide analog.

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

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

7. The composition of claim 6, wherein the sequencing system comprises a zero mode waveguide, an optical confinement, or an observation volume.

8. The composition of claim 1, wherein the modified recombinant polymerase is immobilized on a surface.

9. The composition of claim 1, wherein the modified recombinant polymerase comprises an F526L substitution.

10. The composition of claim 1, wherein the modified recombinant polymerase comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:

1.

11. The composition of claim 1, wherein the modified recombinant polymerase comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:

21.