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

• US 8,420,325 B2
• Filed: 11/04/2010
• Issued: 04/16/2013
• 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): Kim, Young J
• Application Number:
  US12/939,798
• Publication Number:
  US 20110143356A1
• Time in Patent Office: 894 Days
• Field of Search: None
• US Class Current: 435/6.12
• 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. An isolated thermostable Taq DNA polymerase mutant having altered fidelity, wherein said mutant comprises one or more mutated amino acid residues in the active site O-helix of a thermostable polymerase, wherein said mutated amino acid residue is adjacent to an immutable or nearly immutable residue corresponding to Arg659, Lys663, Phe667 or Tyr671 of SEQ ID NO:

2, wherein at least one mutation comprises a residue corresponding to Phe667Leu, and, optionally, additional mutations selected from the group of residues corresponding to;

Asn666Asp;

Asn666Ile;

Ile665Leu;

Leu670Val;

Arg660Tyr;

Arg660Ser;

Gly668Arg;

Arg660Lys;

Gly668Ser;

Gly668Gln;

Thr664Ile and Asn666Asp;

Ala661Ser and Val669Leu;

Ala661Glu, and Ile665Thr; and

Thr664Pro, Ile665Val and Asn666Tyr of SEQ ID NO;

2; and

said Arg659 and Lys663 residues are not mutated.

2. The polymerase mutant of claim 1, wherein said polymerase mutant is a high fidelity mutant.

3. A method for identifying one or more mutations in a gene, comprising amplifying said gene using the high fidelity polymerase mutant of claim 2 under conditions which allow polymerase chain reaction amplification.

4. A method for accurately copying repetitive nucleotide sequences, comprising amplifying said repetitive nucleotide sequence using said high fidelity polymerase mutant of claim 2.

5. A method for diagnosing a genetic disease, comprising amplifying a gene using said high fidelity polymerase mutant of claim 2.

6. A method for identifying one or more mutations in a gene, comprising amplifying said gene using a high fidelity polymerase mutant of claim 1 under conditions which allow polymerase chain reaction amplification.

7. The method of claim 6, wherein said gene is amplified by exposing the strands of said gene to repeated cycles of denaturing, annealing and elongation to produce an amplified product.

8. The method of claim 7, further comprising determining the presence or absence of one or more mutations in the sequence of said gene.

9. A method for accurately copying repetitive nucleotide sequences, comprising amplifying said repetitive nucleotide sequence using a high fidelity polymerase mutant of claim 1.

10. The method of claim 9, wherein said repetitive nucleotide sequence is in a gene.

11. The method of claim 9, wherein said repetitive nucleotide sequence is in a microsatellite between genes.

12. A method for determining an inherited mutation, comprising amplifying a gene using a high fidelity polymerase mutant of claim 1.

13. A method for diagnosing a genetic disease, comprising correlating the inherited mutation determined in claim 12 with said genetic disease.

14. A method for diagnosing a genetic disease, comprising amplifying a gene using a high fidelity polymerase mutant of claim 1.

15. The method of claim 14, wherein said genetic disease comprises mutations in microsatellite or repetitive DNA.

16. The method of claim 15, wherein said genetic disease is cancer.

17. A method for determining the prognosis of a genetic disease, comprising amplifying a gene using a high fidelity polymerase mutant of claim 1.

18. An isolated thermostable polymerase comprising one or more mutated amino acid residues relative to a parent polymerase, wherein the mutant polymerase has increased ability to terminate polymerization when it encounters a template nucleotide complementary to a nucleoside triphosphate which is not present wherein at least one mutation comprises a residue corresponding to Phe667Leu, and, optionally, additional mutations are selected from the group of residues corresponding to:

Asn666Asp;

Asn666Ile;

Ile665Leu;

Leu670Val;

Arg660Tyr;

Arg660Ser;

Gly668Arg;

Arg660Lys;

Gly668Ser;

Gly668Gln;

Thr664Ile and Asn666Asp;

Ala661Ser and Val669Leu;

Ala661Glu and Ile665Thr; and

Thr664Pro, Ile665Val and Asn666Tyr of SEQ ID NO;

2.

19. The polymerase of claim 18, wherein the parent polymerase is selected from the group consisting of:

A DNA polymerase;

a DNA-dependent RNA polymerase;

a reverse transcriptase;

and a Taq polymerase.

20. The polymerase of claim 18, wherein said parent polymerase is Taq polymerase of SEQ ID NO:

2 or a polymerase with structurally similar domains as defined by a crystallographic molecular model.

21. The polymerase of claim 18, wherein said mutant polymerase is generated using random mutagenesis of said parent polymerase.

22. A reaction mixture comprising the polymerase of claim 18.

23. The reaction mixture of claim 22, further comprising at least one oligonucleotide primer.

24. The reaction mixture of claim 23, wherein said oligonucleotide primer is specific for a target associated with a genetic disease.

25. The reaction mixture of claim 24, wherein the target is a repetitive DNA sequence.

26. The reaction mixture of claim 24, wherein the target is a microsatellite.

27. The reaction mixture of claim 24, wherein the target involves a mutation selected from the group consisting of:

a point mutation, an insertion, and a deletion.

28. The reaction mixture of claim 22, wherein said reaction is for a polymerase chain reaction (PCR).