THERMOSTABLE POLYMERASES HAVING ALTERED FIDELITY AND METHODS OF IDENTIFYING AND USING SAME
First Claim
1. A method for identifying a thermostable polymerase having altered fidelity, comprising generating a random population of polymerase mutants by mutating at least one amino acid residue of a thermostable polymerase and screening said population for one or more active polymerase mutants by genetic selection.
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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.
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Citations
50 Claims
- 1. A method for identifying a thermostable polymerase having altered fidelity, comprising generating a random population of polymerase mutants by mutating at least one amino acid residue of a thermostable polymerase and screening said population for one or more active polymerase mutants by genetic selection.
- 9. A method for identifying a thermostable polymerase having altered fidelity, comprising generating a random population of polymerase mutants by mutating at least one amino acid residue in an active site O-helix of a thermostable polymerase and screening said population for one or more active polymerase mutants.
- 16. An isolated thermostable 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.
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27. An isolated nucleic acid molecule encoding a polymerase mutant having high fidelity, comprising a nucleotide sequence encoding substantially an amino acid sequence of Taq DNA polymerase I comprising 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.
- Asn666Asp;
- 28. An isolated nucleic acid molecule encoding a polymerase mutant having low fidelity, comprising a nucleotide sequence encoding substantially an amino acid sequence of Taq DNA polymerase I comprising substitution of one or more amino acids selected from the group consisting of Ala661, Thr664, Asn666 and Leu670.
- 30. A method for identifying one or more mutations in a gene, comprising amplifying said gene using a high fidelity polymerase mutant under conditions which allow polymerase chain reaction amplification.
- 35. A method for accurately copying repetitive nucleotide sequences, comprising amplifying said repetitive nucleotide sequence using a high fidelity polymerase mutant.
- 39. A method for determining an inherited mutation, comprising amplifying a gene using a high fidelity polymerase mutant.
- 41. A method for diagnosing a genetic disease, comprising amplifying a gene using a high fidelity polymerase mutant.
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47. A method for randomly mutagenizing a gene, comprising amplifying said gene using a low fidelity polymerase mutant.
Specification