Thermophilic dna polymerase mutants

Abstract

This disclosure relates to thermophilic family B DNA polymerases comprising a neutral amino acid residue at a certain position near the C-terminus of the catalytic domain, which corresponds to a position occupied by a basic amino acid residue in wild-type Pfu polymerase. The thermophilic family B DNA polymerases provided herein also comprise an N-terminal domain comprising a uracil-binding pocket that has been modified to reduce template uracil binding. Related uses, methods, and compositions are also provided. In some embodiments, the polymerases comprise a 3′-5′ exonuclease domain and/or a sequence nonspecific dsDNA binding domain.

What is claimed is: 
     
         1 - 55 . (canceled) 
     
     
         56 . A nucleic acid comprising a sequence encoding a thermophilic DNA polymerase comprising a family B polymerase N-terminal domain comprising a uracil-binding pocket and a family B polymerase catalytic domain, the family B polymerase N-terminal domain comprising a uracil-binding pocket having an amino acid sequence in which the position corresponding to position 36 of SEQ ID NO: 1 is any amino acid other than P, and the family B polymerase catalytic domain having an amino acid sequence in which the position corresponding to position 762 of SEQ ID NO: 1 is a neutral amino acid residue,
 wherein the thermophilic DNA polymerase comprises an amino acid sequence having at least 90%, 95%, 98%, 99%, or 100% identity to a sequence selected from SEQ ID NOs: 80 to 113 and 127 to 160, wherein X 1  is any amino acid other than P and X 2  is the neutral amino acid residue.   
     
     
         57 . An expression vector comprising the nucleic acid of  claim 56 . 
     
     
         58 . An isolated host cell comprising the expression vector of  claim 57 . 
     
     
         59 . A method of producing a thermophilic DNA polymerase, comprising culturing at least one host cell comprising a the nucleic acid of  claim 56 , wherein the at least one host cell expresses the thermophilic DNA polymerase. 
     
     
         60 . The method of  claim 59 , further comprising isolating the thermophilic DNA polymerase. 
     
     
         61 . A composition comprising the nucleic acid of  claim 56 . 
     
     
         62 . The composition of  claim 61 , comprising at least one hot start inhibitor. 
     
     
         63 . The composition of  claim 61 , comprising at least two hot start inhibitors. 
     
     
         64 . The composition of  claim 62 , wherein each hot start inhibitor is independently selected from an antibody, an Affibody®, a chemical modification, and an oligonucleotide. 
     
     
         65 . The composition of  claim 63 , wherein the composition comprises at least two antibodies. 
     
     
         66 . The composition of  claim 63 , wherein the composition comprises an antibody and an oligonucleotide. 
     
     
         67 - 104 . (canceled) 
     
     
         105 . An isolated host cell comprising the nucleic acid of  claim 56 . 
     
     
         106 . The composition of  claim 63 , wherein each hot start inhibitor is independently selected from an antibody, an Affibody®, a chemical modification, and an oligonucleotide. 
     
     
         107 . The composition of  claim 64 , wherein the composition comprises at least two antibodies. 
     
     
         108 . The composition of  claim 64 , wherein the composition comprises an antibody and an oligonucleotide.