nucleic acid polymerases

• US 8,999,689 B2
• Filed: 02/19/2013
• Issued: 04/07/2015
• Est. Priority Date: 11/30/2001
• Status: Active Grant

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Inventors: Rozzelle, James, Bolchakova, Elena
• Primary Examiner(s): NAVARRO, ALBERT MARK
• Application Number:
  US13/770,252
• Publication Number:
  US 20140038187A1
• Time in Patent Office: 777 Days
• Field of Search: None
• US Class Current: 435/183
• 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 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Y 207/07007 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: Y10S 435/975 Kit

Abstract

The invention provides novel nucleic acid polymerases from strains GK24 and RQ-1 of Thermus thermophilus, and nucleic acids encoding those polymerases, as well as methods for using the polymerases and nucleic acids.

Claims

1. An isolated mutant nucleic acid polymerase comprising SEQ ID NO:

20.

2. The isolated mutant nucleic acid polymerase of claim 1, wherein the polymerase exhibits decreased 5-3′

exonuclease activity relative to its corresponding wild type nucleic acid polymerase.

3. The isolated mutant nucleic acid polymerase of claim 1, wherein the polymerase exhibits reduced discrimination against dideoxynucleotide triphosphates relative to its corresponding wild type nucleic acid polymerase.

4. A kit comprising a container containing the mutant nucleic acid polymerase of claim 1.

5. The kit of claim 4 further comprising a container containing an unlabeled nucleotide, a labeled nucleotide, a balanced mixture of nucleotides, a chain terminating nucleotide, a nucleotide analog, a buffer solution, a solution containing magnesium, a cloning vector, a restriction endonuclease, a sequencing primer, a solution containing reverse transcriptase, or a DNA or RNA amplification primer.

6. The kit of claim 4, adapted for performing DNA sequencing, DNA amplification, reverse transcription, RNA amplification or primer extension.

7. The isolated mutant nucleic acid polymerase of claim 1, wherein the polymerase comprises an amino acid substitution at position 46.

8. The isolated mutant nucleic acid polymerase of claim 7, wherein the amino acid substitution at position 46 comprises a substitution with Asp.

9. The isolated mutant nucleic acid polymerase of claim 1, wherein the polymerase is made by incubating a host cell under conditions sufficient for RNA transcription and translation of a nucleic acid encoding the polymerase operably linked to a promoter.