Dna polymerase mutants having enhanced template discrimination activity

Abstract

This invention relates to mutant Taq DNA polymerase having an enhanced template discrimination activity compared with an unmodified Taq DNA polymerase of SEQ ID NO.:1, wherein the amino acid sequence of the mutant Taq DNA polymerase consists of substitutions at residue positions 783, 784, or a combination of 783 and 784 of the unmodified Taq DNA polymerase of SEQ ID NO.:1.

What is claimed is: 
     
         1 . A mutant Taq DNA polymerase having an enhanced template discrimination activity compared with an unmodified Taq DNA polymerase of SEQ ID NO.:1, wherein the amino acid sequence of the mutant Taq DNA polymerase consists of substitutions at residue positions 783, 784, or a combination of 783 and 784 of the unmodified Taq DNA polymerase of SEQ ID NO.:1. 
     
     
         2 . The mutant Taq DNA polymerase of  claim 1 , wherein the enhanced template discrimination activity comprises at least one property selected from the group consisting of enhanced 3′-mismatch discrimination and enhanced 3′-nucleotide discrimination. 
     
     
         3 . The mutant Taq DNA polymerase of  claim 1 , further comprising polymerase activity at least comparable to about 0.01-fold the polymerase activity of unmodified Taq DNA polymerase. 
     
     
         4 . The mutant Taq DNA polymerase of  claim 1 , wherein the enhanced template discrimination activity comprises enhanced 3′-mismatch discrimination, wherein an average ΔΔCq is at least about 1.0 when the mutant Taq DNA polymerase is evaluated for enhanced 3′-mismatch discrimination by allele-specific PCR assay. 
     
     
         5 . The mutant Taq DNA polymerase of  claim 1 , wherein the enhanced template discrimination activity comprises enhanced 3′-nucleotide discrimination, wherein an average ΔΔCq is at least about 1.0 when the mutant Taq DNA polymerase is evaluated for enhanced 3′-nucleotide discrimination by quantitative PCR. 
     
     
         6 . The mutant Taq DNA polymerase of  claim 1 , wherein the enhanced template discrimination activity comprises enhanced 3′-mismatch discrimination by rhPCR, wherein an average ΔΔCq is at least about 1.0 when the mutant Taq DNA polymerase is evaluated for enhanced 3′-mismatch discrimination by rhPCR with an RDDDDx blocked-cleavable rhPCR primer. 
     
     
         7 . The mutant Taq DNA polymerase of  claim 1 , wherein the enhanced template discrimination activity comprises enhanced 3′-mismatch discrimination by rhPCR, wherein an average ΔΔCq is at least about 0.50 when the mutant Taq DNA polymerase is evaluated for enhanced 3′-mismatch discrimination by rhPCR with an RDxxD blocked-cleavable rhPCR primer. 
     
     
         8 . The mutant Taq DNA polymerase of  claim 1 , wherein the enhanced template discrimination activity comprises enhanced 3′-mismatch discrimination by rhPCR SNP discrimination assay, wherein an average ΔΔCq is at least about 1.0 when the mutant Taq DNA polymerase is evaluated for enhanced 3′-mismatch discrimination by rhPCR SNP discrimination assay of the SMAD7 gene NM_005904, C/T SNP, rs4939827) with RDDDDx blocked-cleavable rhPCR primers consisting of SEQ ID NOs:76 and 77. 
     
     
         9 . The mutant Taq DNA polymerase of  claim 1 , wherein the enhanced template discrimination activity comprises enhanced 3′-mismatch discrimination by rhPCR SNP discrimination assay, wherein an average ΔΔCq is at least about 1.0 when the mutant Taq DNA polymerase is evaluated for enhanced 3′-mismatch discrimination by rhPCR SNP discrimination assay of the SMAD7 gene NM_005904, C/T SNP, rs4939827) with RDxxD blocked-cleavable rhPCR primers consisting of SEQ ID NOs:78 and 79. 
     
     
         10 . The mutant Taq DNA polymerase of  claim 1 , wherein the enhanced template discrimination activity comprises enhanced 3′-mismatch discrimination by rhPCR SNP discrimination assay, wherein an average ΔΔCq is at least about 5.0 when the mutant Taq DNA polymerase is evaluated for enhanced 3′-mismatch discrimination by rhPCR SNP discrimination assay of the SMAD7 gene NM_005904, C/T SNP, rs4939827) with RDxxD blocked-cleavable rhPCR primers consisting of SEQ ID NOs:78 and 79. 
     
     
         11 . The mutant Taq DNA polymerase of  claim 1 , wherein the enhanced template discrimination activity comprises enhanced rare allele discrimination, wherein the enhanced rare allele discrimination is at least 1:10,000 when the mutant Taq DNA polymerase is evaluated by rhPCR SNP discrimination assay of the SMAD7 gene NM_005904, C/T SNP, rs4939827) having a ΔCq of at least 3.0 with RDxxD blocked-cleavable rhPCR primers consisting of SEQ ID NOs:78 and 79. 
     
     
         12 . A mutant Taq DNA polymerase having an enhanced template discrimination activity compared with an unmodified Taq DNA polymerase, wherein the amino acid sequence of the mutant Taq DNA polymerase is selected from the group of the following selected substitutions: V783F, H784Q, or a combination of V783L and H784Q. 
     
     
         13 . The mutant Taq DNA polymerase of  claim 12 , wherein the mutant Taq DNA polymerase has at least 80% sequence identity to one of SEQ ID NOS: 83, 85, 87 or 89. 
     
     
         14 . The mutant Taq DNA polymerase of  claim 12 , wherein the enhanced template discrimination activity comprises at least one property selected from the group consisting of enhanced 3′-mismatch discrimination and enhanced 3′-nucleotide discrimination. 
     
     
         15 . The mutant Taq DNA polymerase of  claim 12 , further comprising polymerase activity at least comparable to about 0.02-fold the polymerase activity of unmodified Taq DNA polymerase. 
     
     
         16 . The mutant Taq DNA polymerase of  claim 12 , wherein the enhanced template discrimination activity comprises enhanced 3′-mismatch discrimination, wherein an average ΔΔCq is at least about 5.0 when the mutant Taq DNA polymerase is evaluated for enhanced 3′-mismatch discrimination by allele-specific PCR assay. 
     
     
         17 . The mutant Taq DNA polymerase of  claim 12 , wherein the enhanced template discrimination activity comprises enhanced 3′-nucleotide discrimination, wherein an average ΔΔCq is at least about 3.0 when the mutant Taq DNA polymerase is evaluated for enhanced 3′-nucleotide discrimination by quantitative PCR. 
     
     
         18 . The mutant Taq DNA polymerase of  claim 12 , wherein the enhanced template discrimination activity comprises enhanced 3′-mismatch discrimination, wherein an average ΔΔCq is at least about 1.0 when the mutant Taq DNA polymerase is evaluated for enhanced 3′-mismatch discrimination by rhPCR with an RDDDDx blocked-cleavable rhPCR primer. 
     
     
         19 . The mutant Taq DNA polymerase of  claim 12 , wherein the enhanced template discrimination activity comprises enhanced 3′-mismatch discrimination, wherein an average ΔΔCq is at least about 0.60 when the mutant Taq DNA polymerase is evaluated for enhanced 3′-mismatch discrimination by rhPCR with an RDxxD blocked-cleavable rhPCR primer. 
     
     
         20 . The mutant Taq DNA polymerase of  claim 12 , wherein the enhanced template discrimination activity comprises enhanced 3′-mismatch discrimination by rhPCR SNP discrimination assay, wherein an average ΔΔCq is at least about 1.0 when the mutant Taq DNA polymerase is evaluated for enhanced 3′-mismatch discrimination by rhPCR SNP discrimination assay of the SMAD7 gene NM_005904, C/T SNP, rs4939827) with RDDDDx blocked-cleavable rhPCR primers consisting of SEQ ID NOs:76 and 77. 
     
     
         21 . The mutant Taq DNA polymerase of  claim 12 , wherein the enhanced template discrimination activity comprises enhanced 3′-mismatch discrimination by rhPCR SNP discrimination assay, wherein an ΔΔCq is at least about 3.5 when the mutant Taq DNA polymerase is evaluated for enhanced 3′-mismatch discrimination by rhPCR SNP discrimination assay of the SMAD7 gene NM_005904, C/T SNP, rs4939827) with RDxxD blocked-cleavable rhPCR primers consisting of SEQ ID NOs:78 and 79. 
     
     
         22 . The mutant Taq DNA polymerase of  claim 12 , wherein the enhanced template discrimination activity comprises enhanced 3′-mismatch discrimination by rhPCR SNP discrimination assay, wherein an ΔΔCq is at least about 15.0 when the mutant Taq DNA polymerase is evaluated for enhanced 3′-mismatch discrimination by rhPCR SNP discrimination assay of the SMAD7 gene NM_005904, C/T SNP, rs4939827) with RDxxD blocked-cleavable rhPCR primers consisting of SEQ ID NOs:78 and 79. 
     
     
         23 . The mutant Taq DNA polymerase of  claim 12 , wherein the enhanced template discrimination activity comprises enhanced rare allele discrimination, wherein the enhanced rare allele discrimination is at least 1:10,000 when the mutant Taq DNA polymerase is evaluated by rhPCR SNP discrimination assay of the SMAD7 gene NM_005904, C/T SNP, rs4939827) having a ΔCq of at least 3.0 with RDxxD blocked-cleavable rhPCR primers consisting of SEQ ID NOs:78 and 79. 
     
     
         24 . A kit for producing an extended primer, comprising: at least one container providing a mutant DNA polymerase according to  claim 1 . 
     
     
         25 . The kit according to  claim 24 , further comprising one or more additional containers selected from the group consisting of: (a) a container providing a primer hybridizable, under primer extension conditions, to a predetermined polynucleotide template; (b) a container providing nucleoside triphosphates; and (c) a container providing a buffer suitable for primer extension. 
     
     
         26 . The kit according to  claim 24 , further comprising one or more additional containers selected from the group consisting of (a) a container containing a blocked-cleavable primer and (b) a container containing RNase H2. 
     
     
         27 . A reaction mixture comprising a mutant DNA polymerase according to  claim 1 , at least one primer, a polynucleotide template, and nucleoside triphosphates. 
     
     
         28 . The reaction mixture according to  claim 27 , wherein the at least one primer comprises a blocked-cleavable primer. 
     
     
         29 . The reaction mixture according to  claim 27 , further comprising RNase H2. 
     
     
         30 . A method for performing rhPCR, comprising performing primer extension with a mutant DNA polymerase of  claim 1 . 
     
     
         31 . A method for performing rhPCR, comprising performing primer extension with a mutant Taq DNA polymerase of  claim 12 . 
     
     
         32 . A mutant Taq DNA polymerase consisting of an amino acid sequence selected from a group consisting of SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:89, SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:155, SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:157, SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178, and SEQ ID NO: 180.