Thermostable polymerases having altered fidelity and methods of identifying and using same
View Patent ↗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.
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).