Recombinant polymerases for improved single molecule sequencing

• US 8,999,676 B2
• Filed: 07/05/2011
• Issued: 04/07/2015
• 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: Emig, Robin, Jia, Lei, Kamtekar, Satwik, Miller, Erik, Cutcliffe, Colleen, Lee, Walter
• Primary Examiner(s): HUTSON, RICHARD G
• Application Number:
  US13/176,697
• Publication Number:
  US 20120034602A1
• Time in Patent Office: 1,372 Days
• Field of Search: None
• US Class Current: 435/91.2
• CPC Class Codes: C12N 9/1241 Nucleotidyltransferases (2.7.7)
• 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 1/6869 Methods for sequencing

Abstract

Provided are compositions comprising recombinant DNA polymerases that include amino acid substitutions, insertions, deletions, and/or exogenous features that confer modified properties upon the polymerase for enhanced single molecule sequencing. Such properties can include enhanced metal ion coordination, reduced exonuclease activity, reduced reaction rates at one or more steps of the polymerase kinetic cycle, decreased branching fraction, altered cofactor selectivity, increased yield, increased thermostability, increased accuracy, increased speed, increased readlength, and the like. Also provided are nucleic acids which encode the polymerases with the aforementioned phenotypes, as well as methods of using such polymerases to make a DNA or to sequence a DNA template.

Claims

1. A composition comprising a recombinant DNA polymerase, which recombinant DNA polymerase comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:

1 or SEQ ID NO;

2;

which recombinant polymerase comprises a mutation at position V250 and a mutation at position L253, wherein identification of positions is relative to wild-type Φ

29 polymerase (SEQ ID NO;

1); and

which recombinant DNA polymerase exhibits polymerase activity.

2. The composition of claim 1, wherein the mutation at position V250 comprises a V250I substitution and the mutation at position L253 comprises an L253A substitution.

3. The composition of claim 1, wherein the composition comprises Mg⁺⁺, wherein the composition is present in a DNA sequencing system that comprises a zero-mode waveguide, and wherein the recombinant polymerase is immobilized on a surface of the zero-mode waveguide in an active form.

4. The composition of claim 1, wherein the mutation at position V250 comprises a V250I substitution.

5. The composition of claim 1, wherein the recombinant polymerase comprises a combination of mutations selected from the group consisting of:

a) V250I, L253A, E375Y, and K512Y; and

b) V250I, L253A, E375Y, A484E, and K512Y.

6. The composition of claim 1, wherein the recombinant polymerase comprises one or more exogenous features at the C-terminal and/or N-terminal region of the polymerase.

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

8. A method of sequencing a DNA template, the method comprising:

a) providing a reaction mixture comprising;

the DNA template,

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

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

one or more nucleotides and/or nucleotide analogs;

b) subjecting the reaction mixture to a polymerization reaction in which the modified recombinant 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; and

c) identifying a time sequence of incorporation of the one or more nucleotides and/or nucleotide analogs into the resulting DNA.