Modified DNA polymerases
The present invention provides, among other things, modified DNA polymerases containing amino acid alterations based on mutations identified in directed evolution experiments designed to select enzymes that are better suited for applications in recombinant DNA technologies.
1. A modified DNA polymerase having a DNA polymerase activity comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence as set forth in SEQ ID NO: 16, or the amino acid sequence as set forth in, which amino acid sequence includes one or more amino acid substitutions relative to SEQ ID NO:16, such substitutions being selected from the group consisting of F752Y, F591L, F591I, E668V, G638R, G638V, E734K, E377K, T609I, T609A, P454S, E582K or G715R of SEQ ID NO: 16 and combinations thereof, wherein the one or more amino acid substitutions alter enzyme activity, fidelity, processivity, elongation rate, stability, or solubility.
2. A modified DNA polymerase having a DNA polymerase activity comprising the amino acid sequence as set forth in SEQ ID NO: 16 with one or more amino acid substitutions selected from the group consisting of F752Y, F591L, F591I, G638V, G638R, E668V, E734K, V356M, E738G, E386K, W772R, or E377K of SEQ ID NO: 16 and combinations thereof, wherein the one or more amino acid substitutions increase the enzyme activity of the DNA polymerase.
3. A modified DNA polymerase having a DNA polymerase activity comprising the amino acid sequence as set forth in SEQ ID NO: 16 with one or more amino acid substitutions selected from the group consisting of F591I, F591L, A550V, E377K, A494V, E734K, G638V, G638R, E668V, D346G, V356M or E738G of SEQ ID NO: 16 and combinations thereof, wherein the one or more amino acid substitutions increase the DNA binding affinity of the DNA polymerase.
4. A modified DNA polymerase having a DNA polymerase activity comprising the amino acid sequence as set forth in SEQ ID NO: 16 with one or more amino acid substitutions selected from the group consisting of R410H, E582K, E652K, A679T, S376G, or T680I of SEQ ID NO: 16 and combinations thereof, wherein the one or more amino acid substitutions decreases the DNA binding affinity of the DNA polymerase.
5. A modified DNA polymerase having a DNA polymerase activity comprising the amino acid sequence as set forth in SEQ ID NO: 16 with one or more amino acid substitutions selected from the group consisting of F591L, F752Y, F591I, E668V, V441I, G638R, S376G or T6801 of SEQ ID NO: 16 and combinations thereof, wherein the one or more amino acid substitutions decreases the fidelity of the DNA polymerase.
6. The modified DNA polymerase of any one of claim 1 , 2 , 3 , 4 or 5 , wherein the DNA polymerase is a fusion polymerase.
7. A kit comprising the modified DNA polymerase of any one of claim 1 , 2 , 3 , 4 or 5 .