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

• US 6,982,144 B2
• Filed: 10/04/2001
• Issued: 01/03/2006
• Est. Priority Date: 11/27/1996
• Status: Expired due to Fees

• Current Assignee: University of Washington
• Original Assignee: University of Washington
• Inventors: Hood, Leroy, Loeb, Lawrence A., Suzuki, Motoshi
• Primary Examiner(s): FREDMAN, JEFFREY NORMAN
• Application Number:
  US09/972,834
• Publication Number:
  US 20020192663A1
• Time in Patent Office: 1,552 Days
• Field of Search: 435/6, 435/193, 530/350, 536/23.1
• US Class Current: 435/5
• 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 Phe667Leu;

Asn666Asp;

Asn666Ile;

Ile665Leu;

Leu670Val;

Arg660Tyr;

Arg660Ser;

Gly668Arg;

Arg660Lys;

Gly668Ser;

Gly668Gln;

Thr664Ile and Asn666Asp;

Ala661Ser and Val669Leu;

Ala661Glu, Ile665Thr, and Phe667Leu; and

Thr664Pro, Ile665Val and Asn666Tyr, and a template neucleic acid molecule.

2. The composition of claim 1, wherein said polymerase mutant comprises the amino acid substitution Phe667Leu.

3. The composition of claim 1, wherein said polymerase mutant comprises the amino acid substitution Asn666Asp.

4. The composition of claim 1, wherein said polymerase mutant comprises the amino acid substitution Asn666Ile.

5. The composition of claim 1, wherein said polymerase mutant comprises the amino acid substitution Ile665Leu.

6. The composition of claim 1, wherein said polymerase mutant comprises the amino acid substitution Leu670Val.

7. The composition of claim 1, wherein said polymerase mutant comprises the amino acid substitution Arg660Tyr.

8. The composition of claim 1, wherein said polymerase mutant comprises the amino acid substitution Arg660Ser.

9. The composition of claim 1, wherein said polymerase mutant comprises the amino acid substitution Gly668Arg.

10. The composition of claim 1, wherein said polymerase mutant comprises the amino acid substitution Arg660Lys.

11. The composition of claim 1, wherein said polymerase mutant comprises the amino acid substitution Gly668Ser.

12. The composition of claim 1, wherein said polymerase mutant comprises the amino acid substitution Gly668Gln.

13. The polymerase mutant of claim 1, wherein said polymerase mutant comprises the amino acid substitutions Thr664Ile and Asn666Asp.

14. The polymerase mutant of claim 1, wherein said polymerase mutant comprises the amino acid substitutions Ala661Ser and Val669Leu.

15. The polymerase mutant of claim 1, wherein said polymerase mutant comprises the amino acid substitutions Ala661Glu, Ile665Thr, and Phe667Leu.

16. The polymerase mutant of claim 1, wherein said polymerase mutant comprises the amino acid substitutions Thr664Pro, Ile665Val and Asn666Tyr.