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 . 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. 
     
     
         2 . The method of  claim 1 , wherein two or more amino-acid residues of said thermostable polymerase are mutated. 
     
     
         3 . The method of  claim 1 , further comprising determining a fidelity of said active polymerase mutant. 
     
     
         4 . The method of  claim 1 , wherein said mutated amino acid residue is adjacent to an immutable or nearly immutable residue. 
     
     
         5 . The method of  claim 4 , wherein said mutated amino acid residue is immediately adjacent to an immutable or nearly immutable residue. 
     
     
         6 . The method of  claim 1 , wherein said mutated amino acid residue is in an O-helix of a thermostable polymerase. 
     
     
         7 . The method of  claim 4 , wherein said mutated-amino acid residue is adjacent to an amino acid residue corresponding to Arg659, Lys663, Phe667 or Tyr671 in Taq DNA polymerase. 
     
     
         8 . The method of  claim 7 , wherein said thermostable polymerase is Taq DNA polymerase. 
     
     
         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. 
     
     
         10 . The method of  claim 9 , wherein two or more amino acid residues of said thermostable polymerase is mutated. 
     
     
         11 . The method of  claim 9 , further comprising determining a fidelity of said active polymerase mutant. 
     
     
         12 . The method of  claim 9 , wherein said mutated amino acid residue is adjacent to an immutable or nearly immutable residue. 
     
     
         13 . The method of  claim 12 , wherein said mutated amino acid residue is immediately adjacent to an immutable or nearly immutable residue. 
     
     
         14 . The method of  claim 12 , wherein said one or more amino acid residues is adjacent to an amino acid residue corresponding to Arg659, Lys663, Phe667 or Tyr671 in Taq DNA polymerase. 
     
     
         15 . The method of  claim 14 , wherein said thermostable polymerase is Taq DNA polymerase. 
     
     
         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. 
     
     
         17 . The polymerase mutant of  claim 16 , wherein said polymerase is Taq DNA polymerase. 
     
     
         18 . The polymerase mutant of  claim 16 , wherein said mutated amino acid residue is adjacent to an immutable or nearly immutable residue. 
     
     
         19 . The polymerase mutant of  claim 18 , wherein said mutated amino acid residue is immediately adjacent to an immutable or nearly immutable residue. 
     
     
         20 . The polymerase mutant of  claim 18 , wherein said mutated amino acid residue is adjacent to an amino acid residue corresponding to Arg659, Lys663, Phe667 or Tyr671 in Taq DNA polymerase. 
     
     
         21 . The polymerase mutant of  claim 20 , wherein said polymerase is Taq DNA polymerase. 
     
     
         22 . The polymerase mutant of  claim 17 , wherein said polymerase mutant is a high fidelity mutant. 
     
     
         23 . The polymerase mutant of  claim 22 , 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. 
     
     
         24 . The polymerase mutant of  claim 17 , wherein said polymerase mutant is a low fidelity mutant. 
     
     
         25 . The polymerase mutant of  claim 24 , wherein said polymerase mutant comprises substitution of one or more amino acids selected from the group consisting of Ala661, Thr664, Asn666 and Leu670. 
     
     
         26 . The polymerase mutant of  claim 25 , wherein said polymerase mutant comprises one or more amino acid substitutions selected from the group consisting of Ala661Glu; Ala661Pro; Thr664Pro; Thr664Asn; Thr664Arg; Asn666Val; Thr664Pro and Val669Ile; Arg660Pro and Leu670Thr; Arg660Trp and Thr664Lys; Ala662Gly and Thr664Asn; Ala661Gly and Asn666Ile; Ala661Pro and Asn666Ile; and Ala661Ser, Ala662Gly, Thr664Ser and Asn666Ile. 
     
     
         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. 
     
     
         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. 
     
     
         29 . The nucleic acid molecule of  claim 28 , wherein said polymerase mutant comprises one or more amino acid substitutions selected from the group consisting of Ala661Glu; Ala661Pro; Thr664Pro; Thr664Asn; Thr664Arg; Asn666Val; Thr664Pro and Val669Ile; Arg660Pro and Leu670Thr; Arg660Trp and Thr664Lys; Ala662Gly and Thr664Asn; Ala661Gly and Asn666Ile; Ala661Pro and Asn666Ile; and Ala661Ser, Ala662Gly, Thr664Ser and Asn666Ile. 
     
     
         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. 
     
     
         31 . A method for identifying one or more mutations in a gene, comprising amplifying said gene using the high fidelity polymerase mutant of  claim 22  under conditions which allow polymerase chain reaction amplification. 
     
     
         32 . The method of  claim 30 , 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. 
     
     
         33 . The method of  claim 32 , further comprising determining the presence or absence of one or more mutations in the sequence of said gene. 
     
     
         34 . The method of  claim 30 , 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. 
     
     
         35 . A method for accurately copying repetitive nucleotide sequences, comprising amplifying said repetitive nucleotide sequence using a high fidelity polymerase mutant. 
     
     
         36 . The method of  claim 35 , wherein said repetitive nucleotide sequence is in a gene. 
     
     
         37 . The method of  claim 35 , wherein said repetitive nucleotide sequence is in a microsatellite between genes. 
     
     
         38 . A method for accurately copying repetitive nucleotide sequences, comprising amplifying said repetitive nucleotide sequence using said high fidelity polymerase mutant of  claim 22 . 
     
     
         39 . A method for determining an inherited mutation, comprising amplifying a gene using a high fidelity polymerase mutant. 
     
     
         40 . A method for diagnosing a genetic disease, comprising correlating the inherited mutation determined in  claim 39  with said genetic disease. 
     
     
         41 . A method for diagnosing a genetic disease, comprising amplifying a gene using a high fidelity polymerase mutant. 
     
     
         42 . A method for diagnosing a genetic disease, comprising amplifying a gene using said high fidelity polymerase mutant of  claim 22 . 
     
     
         43 . The method of  claim 41 , wherein said genetic disease comprises mutations in microsatellite or repetitive DNA. 
     
     
         44 . The method of  claim 43 , wherein said genetic disease is cancer. 
     
     
         45 . A method for determining the prognosis of a genetic disease, comprising amplifying said gene in  claim 41 . 
     
     
         46 . The method of  claim 41 , 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. 
     
     
         47 . A method for randomly mutagenizing a gene, comprising amplifying said gene using a low fidelity polymerase mutant. 
     
     
         48 . A method for randomly mutagenizing a gene, comprising amplifying said gene using said low fidelity polymerase mutant of  claim 24 . 
     
     
         49 . The method of  claim 48 , wherein said polymerase mutant comprises substitution of one or more amino acid residues selected from the group consisting of Ala661, Thr664, Asn666 and Leu670. 
     
     
         50 . The method of  claim 49 , wherein said polymerase mutant comprises one or more amino acid substitutions selected from the group consisting of Ala661Glu; Ala661Pro; Thr664Pro; Thr664Asn; Thr664Arg; Asn666Val; Thr664Pro and Val669Ile; Arg660Pro and Leu670Thr; Arg660Trp and Thr664Lys; Ala662Gly and Thr664Asn; Ala661Gly and Asn666Ile; Ala661Pro and Asn666Ile; and Ala661Ser, Ala662Gly, Thr664Ser and Asn666Ile.