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

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.

1 .- 13 . (canceled) 
     
     
         14 . 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 is selected from the group of residues corresponding to:
 Phe667Tyr; Arg 660Ser; and Arg660Lys;   
       and, optionally, additional mutations selected from the group of residues corresponding to:
 Phe667Leu; Asn666Asp; Asn666Ile; Ile665Leu; Leu670Val; Arg660Tyr; Arg660Ser; Gly668Arg; Arg660Lys; Gly668Ser; Gly668Gln; Thr664Ile and Asn666Asp; Ala661Ser and Val669Leu; Ala661Glu, and Ile665Thr; Ala661Glu; Ile665Thr; and Phe667Leu; and Thr664Pro, Ile665Val and Asn666Tyr of SEQ ID NO:2; and 
 
       said Arg659 and Lys663 residues are not mutated. 
     
     
         15 . The polymerase mutant of  claim 14 , wherein said polymerase mutant is a high fidelity mutant. 
     
     
         16 . 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; wherein the high fidelity polymerase 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 is selected from the group of residues corresponding to:
 Phe667Tyr; Arg 660Ser; and Arg660Lys;   
       and, optionally, additional mutations selected from the group of residues corresponding to:
 Phe667Leu; Asn666Asp; Asn666Ile; Ile665Leu; Leu670Val; Arg660Tyr; Arg660Ser; Gly668Arg; Arg660Lys; Gly668Ser; Gly668Gln; Thr664Ile and Asn666Asp; Ala661Ser and Val669Leu; Ala661Glu, and Ile665Thr; Ala661Glu; Ile665Thr; and Phe667Leu; and Thr664Pro, Ile665Val and Asn666Tyr of SEQ ID NO:2; and 
 
       said Arg659 and Lys663 residues are not mutated. 
     
     
         17 . The method of  claim 16 , 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. 
     
     
         19 . The method of  claim 17 , further comprising determining the presence or absence of one or more mutations in the sequence of said gene. 
     
     
         20 . A method for accurately copying repetitive nucleotide sequences, comprising amplifying said repetitive nucleotide sequence using a high fidelity polymerase mutant wherein at least one mutation is selected from the group of residues corresponding to:
 Phe667Tyr; Arg 660Ser; and Arg660Lys;   
       and, optionally, additional mutations selected from the group of residues corresponding to:
 Phe667Leu; Asn666Asp; Asn666Ile; Ile665Leu; Leu670Val; Arg660Tyr; Arg660Ser; Gly668Arg; Arg660Lys; Gly668Ser; Gly668Gln; Thr664Ile and Asn666Asp; Ala661Ser and Val669Leu; Ala661Glu, and Ile665Thr; Ala661Glu; Ile665Thr; and Phe667Leu; and Thr664Pro, Ile665Val and Asn666Tyr of SEQ ID NO:2; and 
 
       said Arg659 and Lys663 residues are not mutated. 
     
     
         21 . The method of  claim 20 , wherein said repetitive nucleotide sequence is in a gene. 
     
     
         22 . The method of  claim 20 , wherein said repetitive nucleotide sequence is in a microsatellite between genes. 
     
     
         23 . A method for determining an inherited mutation, comprising amplifying a gene using a high fidelity polymerase mutant wherein at least one mutation is selected from the group of residues corresponding to:
 Phe667Tyr; Arg 660Ser; and Arg660Lys;   
       and, optionally, additional mutations selected from the group of residues corresponding to:
 Phe667Leu; Asn666Asp; Asn666Ile; Ile665Leu; Leu670Val; Arg660Tyr; Arg660Ser; Gly668Arg; Arg660Lys; Gly668Ser; Gly668Gln; Thr664Ile and Asn666Asp; Ala661Ser and Val669Leu; Ala661Glu, and Ile665Thr; Ala661Glu; Ile665Thr; and Phe667Leu; and Thr664Pro, Ile665Val and Asn666Tyr of SEQ ID NO:2; and 
 
       said Arg659 and Lys663 residues are not mutated. 
     
     
         24 . The method of  claim 23 , further comprising correlating the inherited mutation with a genetic disease. 
     
     
         25 . The method of  claim 24 , wherein said genetic disease comprises mutations in microsatellite or repetitive DNA. 
     
     
         26 . The method of  claim 24 , wherein said genetic disease is cancer. 
     
     
         27 . The method of  claim 24 , further comprising determining the prognosis of the genetic disease. 
     
     
         28 . 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 is selected from the group of residues corresponding to:
 Phe667Tyr; Arg 660Ser; and Arg660Lys;   
       and, optionally, additional mutations selected from the group of residues corresponding to:
 Phe667Leu; Asn666Asp; Asn666Ile; Ile665Leu; Leu670Val; Arg660Tyr; Arg660Ser; Gly668Arg; Arg660Lys; Gly668Ser; Gly668Gln; Thr664Ile and Asn666Asp; Ala661Ser and Val669Leu; Ala661Glu, and Ile665Thr; Ala661Glu; Ile665Thr; and Phe667Leu; and Thr664Pro, Ile665Val and Asn666Tyr of SEQ ID NO:2. 
 
     
     
         29 . The polymerase of  claim 28 , 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.   
     
     
         30 . The polymerase of  claim 28 , 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. 
     
     
         31 . The polymerase of  claim 28 , wherein said mutant polymerase is generated using random mutagenesis of said parent polymerase. 
     
     
         32 . The polymerase of  claim 28  in admixture with at least one oligonucleotide primer. 
     
     
         33 . The polymerase of  claim 32 , wherein said oligonucleotide primer is specific for a target associated with a genetic disease. 
     
     
         34 . The polymerase of  claim 33 , wherein the target is a repetitive DNA sequence. 
     
     
         35 . The polymerase of  claim 33 , wherein the target is a microsatellite. 
     
     
         36 . The polymerase of  claim 33 , wherein the target involves a mutation selected from the group consisting of,
 a point mutation, an insertion, and a deletion.