Generation of modified polymerases for improved accuracy in single molecule sequencing

• US 8,257,954 B2
• Filed: 03/30/2009
• Issued: 09/04/2012
• 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: Clark, Sonya, Bibillo, Arek, Peluso, Paul, Christians, Fred, He, Molly, Park, Insil, Lee, Harold, Bjornson, Keith, Jia, Lei, Emig, Robin
• Primary Examiner(s): HUTSON, RICHARD G
• Application Number:
  US12/384,112
• Publication Number:
  US 20100112645A1
• Time in Patent Office: 1,254 Days
• Field of Search: None
• US Class Current: 435/194
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/101 DNA polymerase

Abstract

Provided are compositions comprising modified recombinant polymerases that exhibit branching fractions that are less than the branching fractions of the polymerases from which they were derived, or branching fractions that are less than about 25% for a phosphate-labeled nucleotide analog. Also provided are compositions comprising modified recombinant polymerases that exhibit closed polymerase/DNA complexes with increased stability relative to the parental polymerases. Also provided are compositions comprising modified recombinant polymerases that exhibit decreased rate constants relative to the parental polymerases. Provided are methods for generating polymerases with the aforementioned phenotypes. Provided are methods of using such polymerases to make a DNA or to sequence a DNA template.

Claims

1. A composition comprising a modified recombinant Φ

29-type DNA polymerase, which modified recombinant polymerase comprises an amino acid sequence that is at least 80% identical to SEQ ID NO;

1 and comprises an alanine substitution at position 253, wherein numbering of positions is relative to SEQ ID NO;

1, and which modified recombinant polymerase exhibits polymerase activity.

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

1.

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

29, PZA, or M2Y polymerase.

4. The composition of claim 1, wherein the modified recombinant polymerase is an exonuclease deficient polymerase.

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

6. The composition of claim 5, wherein the nucleotide analog comprises a fluorophore.

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

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

9. The composition of claim 8, wherein the sequencing system comprises a zero mode waveguide.

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

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:

1.

12. A method of making a DNA, the method comprising:

a) providing a reaction mixture comprising;

a template,

a replication initiating moiety that complexes with or is integral to the template,

the modified recombinant DNA polymerase of claim 1, which polymerase is capable of replicating at least a portion of the template using the moiety in a template-dependent polymerase reaction, and

one or more nucleotides and/or nucleotide analogs; and

b) reacting the mixture such that the polymerase replicates at least a portion of the template in a template-dependent manner, whereby the one or more nucleotides and/or nucleotide analogs are incorporated into the resulting DNA.