Thermostable polymerases having altered fidelity and methods of identifying and using same

• US 7,312,059 B2
• Filed: 03/31/2005
• Issued: 12/25/2007
• Est. Priority Date: 11/27/1996
• Status: Expired due to Fees

• Current Assignee: University of Washington
• Original Assignee: University of Washington
• Inventors: Loeb, Lawrence A., Hood, Leroy, Suzuki, Motoshi
• Primary Examiner(s): FREDMAN, JEFFREY NORMAN
• Application Number:
  US11/096,645
• Publication Number:
  US 20050196800A1
• Time in Patent Office: 999 Days
• Field of Search: 435/193
• US Class Current: 435/193
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material

Abstract

The present invention provides a method for identifying a thermostable polymerase having altered fidelity. The method consists of generating a random population of polymerase mutants by mutating at least one amino acid residue of a thermostable polymerase and screening the population for one or more active polymerase mutants by genetic selection. For example, the invention provides a method for identifying a thermostable polymerase having altered fidelity by mutating at least one amino acid residue in an active site O-helix of a thermostable polymerase. The invention also provides thermostable polymerases and nucleic acids encoding thermostable polymerases having altered fidelity, for example, high fidelity polymerases and low fidelity polymerases. The invention additionally provides a method for identifying one or more mutations in a gene by amplifying the gene with a high fidelity polymerase. The invention further provides a method for accurately copying repetitive nucleotide sequences using a high fidelity polymerase mutant. The invention also provides a method for diagnosing a genetic disease using a high fidelity polymerase mutant. The invention further provides a method for randomly mutagenizing a gene by amplifying the gene using a low fidelity polymerase mutant.

Claims

1. A composition comprising a Taq DNA polymerase mutant, wherein said polymerase mutant comprises one or more amino acid substitutions selected from the group consisting of Ala661Glu;

Ala661Pro;

Thr664Pro;

Thr664Asn;

Thr664Arg;

Asn666Val;

Thr664Pro and Val669Ile;

Arg660Pro and Leu670Thr;

Arg660Trp and Thr664Lys;

Ala662Gly and Thr664Asn;

Ala661Gly and Asn666Ile;

Ala661Pro and Asn666Ile; and

Ala661Ser, Ala662Gly, Thr664Ser and Asn666Ile, and a template nucleic acid molecule.

2. The polymerase mutant of claim 1, wherein said polymerase mutant comprises the amino acid substitution Ala661Glu.

3. The polymerase mutant of claim 1, wherein said polymerase mutant comprises the amino acid substitution Ala661Pro.

4. The polymerase mutant of claim 1, wherein said polymerase mutant comprises the amino acid substitution Thr664Pro.

5. The polymerase mutant of claim 1, wherein said polymerase mutant comprises the amino acid substitution Thr664Asn.

6. The polymerase mutant of claim 1, wherein said polymerase mutant comprises the amino acid substitution Thr664Arg.

7. The polymerase mutant of claim 1, wherein said polymerase mutant comprises the amino acid substitution Asn666Val.

8. The polymerase mutant of claim 1, wherein said polymerase mutant comprises the amino acid substitutions Thr664Pro and Val669Ile.

9. The polymerase mutant of claim 1, wherein said polymerase mutant comprises the amino acid substitutions Arg660Pro and Leu670Thr.

10. The polymerase mutant of claim 1, wherein said polymerase mutant comprises the amino acid substitutions Arg660Trp and Thr664Lys.

11. The polymerase mutant of claim 1, wherein said polymerase mutant comprises the amino acid substitutions Ala662Gly and Thr664Asn.

12. The polymerase mutant of claim 1, wherein said polymerase mutant comprises the amino acid substitutions Ala661Gly and Asn666Ile.

13. The polymerase mutant of claim 1, wherein said polymerase mutant comprises the amino acid substitutions Ala661Pro and Asn666Ile.

14. The polymerase mutant of claim 1, wherein said polymerase mutant comprises the amino acid substitutions Ala661Ser, Ala662Gly, Thr664Ser and Asn666Ile.