Mutant RB69 DNA polymerase

• US 8,703,461 B2
• Filed: 08/31/2012
• Issued: 04/22/2014
• Est. Priority Date: 06/05/2009
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Peris, Marian, Phelan, Michael, Rosenblum, Barnett, Hendricks, Stephen
• Primary Examiner(s): Hutson, Richard
• Application Number:
  US13/600,416
• Publication Number:
  US 20130005020A1
• Time in Patent Office: 599 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 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6872 involving mass spectrometry
• CPC Class Codes: C12Q 2537/157 A reaction step characterised by the number of molecules incorporated or released
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2563/137 Metal/ion, e.g. metal label
• CPC Class Codes: C12Q 2565/30 Detection characterised by liberation or release of label
• CPC Class Codes: C12Q 2565/632 being a surface enhanced, e.g. resonance, Raman spectrometer
• CPC Class Codes: C12Y 207/07007 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: G01N 2500/00 Screening for compounds of potential therapeutic value

Abstract

Provided herein are mutant DNA-dependent polymerases which are derived from, or otherwise related to, wild type RB69 DNA polymerase. These mutant polymerases are capable of selectively binding labeled nucleotides. These mutant polymerases are also capable of incorporating a variety of naturally occurring and modified nucleotides, including, for example, terminator nucleotides.

Claims

1. An isolated mutant DNA polymerase comprising the amino acid sequence of SEQ ID NO:

2.

2. The isolated mutant DNA polymerase of claim 1, which binds a labeled nucleotide.

3. The isolated mutant DNA polymerase of claim 1, which incorporates a reversible terminator nucleotide having an azidomethyl blocking group on the 3′

sugar position.

4. The isolated mutant DNA polymerase of claim 1 further comprising a reporter moiety.

5. The isolated mutant DNA polymerase of claim 4, wherein the reporter moiety is an energy transfer donor moiety.

6. The isolated mutant DNA polymerase of claim 5, wherein the energy transfer donor moiety is a fluorescent dye or a nanoparticle.

7. A system comprising a mutant DNA polymerase of claim 1 bound to a DNA template and a primer, and a nucleotide transiently-bound to the mutant DNA polymerase.