Modified type A DNA polymerases

• US 10,457,968 B2
• Filed: 11/03/2009
• Issued: 10/29/2019
• Est. Priority Date: 11/03/2008
• Status: Active Grant

• Current Assignee: Kapa Biosystems Co,
• Original Assignee: Kapa Biosystems, Inc. (Roche Holding AG)
• Inventors: Bourn, William, Appel, Maryke, Rush, Gavin, Foskett, John, McEwan, Paul
• Primary Examiner(s): Hutson, Richard G
• Application Number:
  US13/061,940
• Publication Number:
  US 20110281305A1
• Time in Patent Office: 3,647 Days
• Field of Search: None
• CPC Class Codes: C12N 15/1058 Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms
• 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 1/485 involving kinase
• CPC Class Codes: C12Y 207/07007 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: G01N 2333/9126 DNA-directed DNA polymerase (2.7.7.7)

Abstract

The present invention provides improved DNA polymerases, in particular, type A DNA polymerases, that may be better suited for applications in recombinant DNA technologies. Among other things, the present invention provides modified DNA polymerases derived from directed evolution experiments designed to select mutations that confer advantageous phenotypes under conditions used in industrial or research applications.

Claims

1. A modified Taq DNA polymerase that:

(i) has an amino acid sequence that has at least 95% amino acid sequence identity with wild type Taq DNA polymerase set out in SEQ ID NO.;

1, but includes a substitution relative to SEQ ID NO;

1 at a position corresponding to position 507 of SEQ ID NO.;

1, which substitution is E507K; and

(ii) has polymerase activity in catalyzing DNA template-directed synthesis of a DNA polynucleotide, which activity has increased salt-tolerance relative to the wild type Taq DNA polymerase of SEQ ID NO.;

1.

2. The modified Taq DNA polymerase of claim 1, wherein the polymerase has improved ability relative to that of SEQ ID NO.:

1, when used in a PCR reaction, to amplify DNA fragments of 2 kilobases or more in length.

3. The modified Taq DNA polymerase of claim 1, wherein the polymerase has increased heparin-binding affinity relative to that of SEQ ID NO.:

1.

4. The modified Taq DNA polymerase of claim 1, wherein the amino acid percent identity is at least 96%.

5. The modified Taq DNA polymerase of claim 1, wherein the amino acid percent identity is at least 97%.

6. The modified Taq DNA polymerase of claim 1, wherein the amino acid percent identity is at least 98%.

7. A modified Taq DNA polymerase having the amino acid sequence of SEQ ID NO.:

16 (E7S).