Thermostable polymerases having altered fidelity and methods of identifying and using same
First Claim
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.
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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
16 Claims
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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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16)
- Asn666Asp;
Specification