Polymerase variants for DNA sequencing

• US 20080076123A1
• Filed: 09/27/2006
• Published: 03/27/2008
• Est. Priority Date: 09/27/2006
• Status: Abandoned Application

• Current Assignee: Fluidigm Corporation
• Original Assignee: Helicos Biosciences Corporation
• Inventors: Buzby, Philip Richard
• Application Number:
  US11/528,959
• Publication Number:
  US 20080076123A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12Y 207/07007 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase

Abstract

Described herein is a mutant polymerase, specifically the Klenow exo⁻ polymerase in which the proline at amino acid position 680 is replaced by a glycine (P680G), and its use in sequencing a single polynucleotide template using single molecule sequencing.

Claims

1. A method for determining the sequence of a nucleic acid comprising the steps of;

contacting a nucleic acid duplex, comprising primer nucleic acid hybridized to a template nucleic acid with a Klenow exo⁻

DNA polymerase having a glycine for proline substitution at position 680 in the presence of a first labeled nucleotide under conditions that permit the polymerase to add nucleotides to said primer in a template-dependent manner, detecting a signal from the incorporated labeled nucleotide, and repeating said contacting and detecting steps at least once, wherein sequential detection of incorporated labeled nucleotide determines the sequence of the nucleic acid.

2. The method of claim 1, wherein said duplex is attached to a surface.

3. The method of claim 2, wherein said surface comprises a plurality of duplex immobilized at different positions on the substrate.

4. The method of claim 3, wherein at least some of said duplex are individually optically resolvable.

5. The method of claim 1, wherein said label is a fluorescent label.

6. The method of claim 5, wherein the fluorescent label comprises one or more of Cy3 or Cy4.

7. The method of claim 1, wherein the presence or absence of label is determined with total internal reflection fluorescence (TIRF) microscopy.

8. A Klenow exo⁻

DNA polymerase for single molecule sequencing comprising a glycine for proline substitution at position 680.