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 provide 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.

What is claimed is:  
     
         1 . A DNA polymerase having at least one mutation in the nucleotide label interaction region, wherein the DNA polymerase has reduced discrimination for fluorescein-type dye labeled nucleotides.  
     
     
         2 . A DNA polymerase according to  claim 1 , wherein the mutation is in portion of the nucleotide-label interaction region selected from the group consisting of (i) the O-helix, (ii) the K helix, and (iii) the inter O—P helical loop.  
     
     
         3 . A DNA polymerase according to  claim 2 , wherein the mutation is segment of the enzyme corresponding to amino acid residue selected from the group consisting of E520. A531, L522, R523, E524, A525, H526, P527, I 528, V529, E530, K531, I532, R536, E537, R573, Q582, N583, V586, R587, P589, Q592, R593, R595, D610, T612, Q613, E615, R636, D637, T640, F647, V654, D655, P656, L657, R659, R660, T664, E681, L682, A683, I684, P685, E688, F692, Q754, H784, L817, E820, L828, K831, and E832.  
     
     
         4 . A DNA polymerase according to  claim 3 , wherein the mutation is at a position selected from the group consisting of, R595, D655, R660, T664 and E681.  
     
     
         5 . A DNA polymerase according to  claim 4 , wherein the DNA polymerase is Taq DNA polymerase.  
     
     
         6 . A DNA polymerase according to  claim 5 , wherein the mutation is selected from the group consisting of R660D, D655L, E618G, and R595E.  
     
     
         7 . A DNA polymerase according to  claim 6 , wherein comprising 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).  
     
     
         8 . A DNA polymerase according to  claim 2 , wherein the DNA polymerase is a thermostable DNA polymerase.  
     
     
         9 . A polynucleotide encoding a DNA polymerase according to  claim 1 .  
     
     
         10 . An expression vector having a promoter, wherein the vector comprises a polynucleotide according to  claim 1  in functional combination with the promoter.  
     
     
         11 . A host cell comprising an expression vector according to  claim 10 .  
     
     
         12 . A method of synthesizing a fluorescently labeled polynucleotide, said method comprising the step of mixing a DNA polymerase according to  claim 1  with a primed template.  
     
     
         13 . A method according to  claim 12 , wherein the primed template is a primed template in a chain termination sequencing reaction.  
     
     
         14 . A method according to  claim 12 , wherein the primed template is a primed template in a polymerase chain reaction.  
     
     
         15 . A kit for fluorescently labeling a labeling a polynucleotide, the kit comprising a DNA polymerase according to  claim 1  and a fluorescently labeled nucleotide.