Mutant RB69 DNA polymerase

• US 9,399,767 B2
• Filed: 02/27/2014
• Issued: 07/26/2016
• 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 G
• Application Number:
  US14/191,997
• Publication Number:
  US 20140234940A1
• Time in Patent Office: 880 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: 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. A system comprising a mutant DNA polymerase bound to a DNA template and a primer, and a nucleotide transiently-bound to the mutant DNA polymerase, wherein the mutant DNA polymerase comprises the amino acid of SEQ ID NO:

2.

2. The system of claim 1, further comprising a metal cation that inhibits incorporation of the transiently-bound nucleotide.

3. The system of claim 2, wherein the metal cation is selected from a group consisting of calcium, scandium, titanium, vanadium, chromium, iron, cobalt, nickel, copper, zinc, gallium, germanium, arsenic, and selenium.

4. The system of claim 1, wherein the transiently-bound nucleotide is linked to a report moiet.

5. The system of claim 1, wherein the mutant DNA polymerase further comprises a reporter moiety.

6. The system of claim 5, wherein the reporter moiety comprises an energy transfer donor moiety.

7. The system of claim 5, wherein the reporter moiety comprises a fluorescent dye or a nanoparticle.

8. The system of claim 1, wherein the transiently-bound nucleotide comprises a reversible terminator nucleotide.

9. The system of claim 8, wherein the reversible terminator nucleotide comprises an azidomethyl blocking group on the 3′

sugar position.