Polymerase variants

Abstract

Described herein is a variant pol6 polymerase having at least one mutation selected from H223, N224, Y225, H227, I295, Y342, T343, I357, S360, L361, I363, S365Q, S366, Y367, P368, D417, E475, Y476, F478, K518, H527, T529, M531, N535, G539, P542, N545, Q546, A547, L549, I550, N552, G553, F558, A596, G603, A610, V615, Y622, C623, D624, I628, Y629, R632, N635, M641, A643, I644, T647, I648, T651, I652, K655, W656, D657, V658, H660, F662, L690 and combinations thereof.

What is claimed is: 
     
         1 . An isolated polypeptide having a DNA polymerase activity comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1, wherein the amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1 comprises at least two amino acid substitutions relative to SEQ ID NO: 2, wherein one of said at least two amino acid substitutions corresponds to a position of SEQ ID NO: 2 selected from the group consisting of H223, N224, Y225, H227, I295, Y342, T343, I357, S360, L361, I363, S365, Y367, P368, D417, E475, Y476, F478, K518, H527, M531, N535, G539, P542, Q546, A547, L549, I550, N552, G553, F558, A596, G603, A610, V615, Y622, C623, D624, I628, Y629, R632, N635, M641, A643, I644, T647, I648, T651, I652, K655, W656, D657, V658, H660, F662, and L690. 
     
     
         2 . The isolated polypeptide of  claim 1 , wherein at least one of the at least two substitutions is selected from the group consisting of H223A, N224Y/L, Y225L/T/I/F/A, H227P, I295W/F/M/E, Y342L/F, T343N/F, I357G/L/Q/H/W/M/A/E/Y/P, S360G, L361M/W/V, I363V, S365Q/W/M/A/G, Y367L/E/M/P/N, P368G, D417P, E475D, Y476V, F478L, K518Q, H527W/R/L, M531H/Y/A/K/R/W/T/L/V, N535L/Y/M/K/I, G539Y/F, P542E/S, Q546W/F, A547M/Y/W/F/V/S, L549Q/Y/H/G/R, I550A/W/T/G/F/S, N552L/M/S, G553S/T, F558P/T, A596S, G603T, A610T/E, V615A/T, Y622A/M, C623G/S/Y, D624F, I628Y/V/F, Y629W/H/M, R632L/C, N635D, M641L/Y, A643L, I644H/M/Y, T647G/A/E/K/S, I648K/R/V/N/T, T651Y/F/M, I652Q/G/S/N/F/T, K655G/F/E/N, W656E, D657R/P/A, V658L, H660A/Y, F662I/L, and L690M. 
     
     
         3 . The isolated polypeptide of  claim 1 , wherein said isolated polypeptide has an altered characteristic as compared to a polymerase comprising SEQ ID NO: 1, wherein said altered characteristic is selected from the group consisting of fidelity, processivity, elongation rate, stability, solubility, and the ability to bind a nucleotide polyphosphate, and/or the ability to incorporate a nucleotide polyphosphate into a growing DNA strand. 
     
     
         4 . The isolated polypeptide of  claim 3 , wherein the nucleotide polyphosphate has 4, 5, 6, 7 or 8 phosphates. 
     
     
         5 . The isolated polypeptide of  claim 4 , wherein the polyphosphate nucleotides are tagged. 
     
     
         6 . The isolated polypeptide of  claim 1 , wherein said isolated polypeptide has a substitution corresponding to E475D. 
     
     
         7 . The isolated polypeptide of  claim 1 , wherein said isolated polypeptide has a substitution corresponding to F478L. 
     
     
         8 . The isolated polypeptide of  claim 1 , wherein said isolated polypeptide has a substitution corresponding to K518Q. 
     
     
         9 . The isolated polypeptide of  claim 1 , wherein said isolated polypeptide has a substitution corresponding to T529M. 
     
     
         10 . The isolated polypeptide of  claim 1 , wherein said isolated polypeptide has a substitution corresponding to N535L. 
     
     
         11 . The isolated polypeptide of  claim 1 , wherein said isolated polypeptide has a substitution corresponding to A547F. 
     
     
         12 . The isolated polypeptide of  claim 1 , wherein said isolated polypeptide has a substitution corresponding to G553S. 
     
     
         13 . The isolated polypeptide of  claim 1 , wherein said isolated polypeptide has a substitution corresponding to T647G. 
     
     
         14 . The isolated polypeptide of  claim 1 , wherein said isolated polypeptide has a substitution corresponding to T651Y. 
     
     
         15 . The isolated polypeptide of  claim 1 , wherein said isolated polypeptide has a substitution corresponding to I652Q. 
     
     
         16 . The isolated polypeptide of  claim 1 , wherein said at least two amino acid substitutions comprises a set of substitutions selected from the group consisting of:
 T651Y+N535L;   Y342L+E475D+F478L;   T343N+D417P+K518Q;   I363V+E475D+Y476V;   T651Y+P542E+Q546W;   T651Y+P542E+S366A;   T647G+A547F+Y225T;   A547F+A610T+Y225I;   T529M+T647G+A547F;   T647G+A547F+T529M;   T529M+A610T+A547F;   M641Y+T529M+A547F;   T647G+C623G+A547F;   A610T+I295W+T651Y;   V615A+M531Y+T647G; and   H223A+G553S+A643L+F662I.   
     
     
         17 . A composition comprising a DNA polymerase attached to an alpha-hemolysin monomer, wherein said DNA polymerase comprises a polypeptide of  claim 1 . 
     
     
         18 . The composition of  claim 18 , wherein the alpha-hemolysin monomer is a part of a heptameric alpha-hemolysin nanopore. 
     
     
         19 . A biochip comprising a nanopore formed in a membrane disposed adjacent to a sensing electrode, wherein said nanopore is attached to a DNA polymerase comprising a polypeptide according to  claim 1 .