Dna polymerases having improved labeled nucleotide incorporation properties

Abstract

The present invention relates to mutant DNA polymerases that exhibit reduced discrimination against labeled nucleotides into polynucleotides. The DNA polymerases of the invention have at least one mutation in the nucleotide label interaction region of the enzyme such the mutation results in reduced discrimination against labeled nucleotides. The nucleotide label interaction regions is located at portions of the O-helix, (ii) the K helix, and (iii) the inter O-P helical loop of Taq DNA polymerase or analogous positions in other DNA polymerases. In addition to providing novel mutant DNA polymerases, the invention also provides polynucleotides encoding the subject mutant DNA polymerases. The polynucleotides provided may comprise expression vectors for the recombinant production of the mutant polymerases. The invention also provides host cells containing the subject polynucleotides. The invention also includes numerous methods of using the subject DNA polymerases, including uses for chain termination sequencing and PCR. Another aspect of the invention is to provide kits for synthesizing fluorescently labeled polynucleotides in accordance with the methods of the invention. Kits of the invention comprise a mutant DNA polymerase of the invention and a fluorescently labeled nucleotide that exhibits reduced discrimination with respect to the mutant DNA polymerase in the kit.

1 - 15 . (canceled) 
     
     
         16 . A method of synthesizing a fluorescently labeled polynucleotide, said method comprising the step of mixing a DNA polymerase with a primed template, wherein the DNA polymerase has at least one mutation as defined with respect to a naturally occurring DNA polymerase, wherein the mutation is at an amino acid residue position selected from the group consisting of E520, A531, L522, R523, E524, A525, H526, P527, 1528, V529, E530, K531, 1532, R536, E537, R573, Q582, N583, V586, R587, P589, Q592, R593, R595, D610, T612, Q613, R636, D637, T640, F647, V654, D655, P656, L657, R659, R660, T664, E681, L682, A683, 1684, P685, E688, F692, Q754, H784, L817, E820, L828, K831, and E832, wherein the position of said amino acid residue within the DNA polymerase is defined with respect to Taq DNA polymerase and wherein the DNA polymerase has at least 2 fold reduced discrimination for a fluorescein-type dye labeled nucleotide as compared with the naturally occurring DNA polymerase. 
     
     
         17 . The method of  claim 16 , wherein the primed template is a primed template in a chain termination sequencing reaction. 
     
     
         18 . The method of  claim 16 , wherein the primed template is a primed template in a polymerase chain reaction. 
     
     
         19 . The method of  claim 16 , wherein the mutation in the DNA polymerase is at a position selected from the group consisting of, R595, D655, R660, T664 and E681. 
     
     
         20 . The method of  claim 19 , wherein the mutation in the DNA polymerase is selected from the group consisting of R660D, D655L, E681G, and R595E. 
     
     
         21 . The method of  claim 20 , wherein the mutation in the DNA polymerase comprises a mutation set belonging to the group consisting of (G46D, R660D, F667Y), (G46D, R595D, R660D, F667Y), and (G46D, R660D, F667Y, E681G), and (G46D, F667Y, E681G). 
     
     
         22 . The method of  claim 16 , wherein the DNA polymerase is a thermostable DNA polymerase. 
     
     
         23 . The method of  claim 22 , wherein the naturally occurring DNA polymerase is Taq DNA polymerase. 
     
     
         24 . The method of  claim 16 , wherein the mutation in the DNA polymerase is at a position selected from the group consisting of, R595, D655, R660, T664 and E681. 
     
     
         25 . The method of  claim 24 , wherein the DNA polymerase is Taq DNA polymerase. 
     
     
         26 . The method of  claim 25 , wherein the mutation in the DNA polymerase is selected from the group consisting of R660D, D655L, E681G, and R595E. 
     
     
         27 . The method of  claim 26 , wherein the mutation in the DNA polymerase comprises a mutation set belonging to the group consisting of (G46D, R660D, F667Y), (G46D, R595D, R660D, F667Y), and (G46D, R660D, F667Y, E681G), and (G46D, F667Y, E681G).