Dna polymerase mutants having enhanced template discrimination activity

Abstract

This invention relates to mutant DNA polymerases having an enhanced template discrimination activity compared with the corresponding unmodified DNA polymerase counterparts, wherein the amino acid sequence of the mutant DNA polymerase includes at least one substitution at residue positions structurally and functionally homologous or orthologous positions 783 or 784 of an unmodified Taq DNA polymerase.

1 . 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 includes at least one substitution at residue positions 783 or 784 of the unmodified Taq DNA polymerase. 
     
     
         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 comprises one of following selected substitutions: (1) A661E; I665W; F667L; (2) V783F; (3) H784Q; or (4) V783L; 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 [corresponding to Taq DNA polymerase mutant including only one of the following (1) A661E; I665W; F667L; (2) V783F; (3) H784Q; or (4) V783L; H784Q]. 
     
     
         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 - 51 . (canceled) 
     
     
         52 . A recombinant nucleic acid encoding the mutant DNA polymerase of  claim 1 . 
     
     
         53 . A method for conducting primer extension, comprising: contacting a mutant DNA polymerase of  claim 1  with a primer, a polynucleotide template, and nucleoside triphosphates under conditions suitable for a primer extension method, thereby producing an extended primer. 
     
     
         54 . The method of  claim 53 , wherein the primer comprises a blocked-cleavable primer. 
     
     
         55 . The method of  claim 54 , further comprising an RNase H2 enzyme. 
     
     
         56 . The method of  claim 55 , wherein the primer extension method comprises a method for conducting polymerase chain reaction (PCR). 
     
     
         57 . The method of  claim 56 , wherein the method for conducting PCR comprises allele-specific PCR. 
     
     
         58 . The method of  claim 56 , wherein the method for conducting PCR comprises detecting a rare allele at a level of discrimination of >1:10,000. 
     
     
         59 . A kit for producing an extended primer, comprising: at least one container providing a mutant DNA polymerase of  claim 1 . 
     
     
         60 . The kit according to  claim 59 , 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. 
     
     
         61 . The kit according to  claim 59 , 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. 
     
     
         62 - 64 . (canceled) 
     
     
         65 . A method for performing rhPCR, comprising performing primer extension with a mutant DNA polymerase of  claim 1 . 
     
     
         66 . (canceled) 
     
     
         67 . A mutant Taq DNA polymerase comprising the amino acid sequence selected from a group consisting of (1) A661E; I665W; F667L [SEQ ID NO:87]; (2) V783F [SEQ ID NO:83]; (3) H784Q [SEQ ID NO:85]; (4) V783L; H784Q [SEQ ID NO:89]; (5) H784A [SEQ ID NO:147]; (6) H784S [SEQ ID NO:149]; (7) H784I [SEQ ID NO:155]; (8) H784T [SEQ ID NO:151], (9) H784V [SEQ ID NO:153]; (10) H784M [SEQ ID NO:157]; (11) H784F [SEQ ID NO:159]; or (12) H784Y [SEQ ID NO:161]. 
     
     
         68 . A mutant Taq DNA polymerase of KlenTaq Polymerase comprising the amino acid sequence selected from a group consisting of (1) A661E; I665W; F667L [SEQ ID NO: 170]; (2) V783F [SEQ ID NO: 172]; (3) H784Q [SEQ ID NO: 174]; (4) V783L; H784Q [SEQ ID NO: 176]; (5) H784S [SEQ ID NO: 178]; or (6) H784Y [SEQ ID NO: 180].