C - Chemistry – Metallurgy – 12 – N
Patent
C - Chemistry, Metallurgy
12
N
C12N 15/54 (2006.01) C12N 9/12 (2006.01) C12P 19/34 (2006.01) C12Q 1/68 (2006.01)
Patent
CA 2243985
A recombinant, thermostable DNA polymerase is provided herein. Such polymerase comprises the amino acid sequence LeuSerXaaXaaLeuXaaXaaProXaaXaaGlu (SEQ ID NO: 1), wherein "Xaa" at positions 3, 6, 9, and 10 of the sequence are any amino acid residue, "Xaa" at position 4 in the sequence is a polar amino acid, or a positively- charged amino acid and "Xaa" at position 7 of the sequence is Val or Ile. The recombinant, thermostable DNA polymerase has a level of discrimination against incorporation of nucleotides which are labeled with fluorescein family dyes which is reduced in comparison to the native form of the polymerase. Such modified recombinant, thermostable DNA polymerases have enhanced efficiency for incorporating unconventional nucleotides, e.g., those labeled with fluorescein family dyes. Such modified recombinant, thermostable DNA polymerases can be prepared by methods of recombinant DNA technology. Such recombinant, thermostable DNA polymerases are advantageous in many in vitro DNA synthesis applications, e.g., DNA sequencing, synthesis of labeled DNA or the production of labeled primer extension products. Such recombinant, thermostable DNA polymerases are particularly useful in chain termination nucleic acid sequencing methods. Nucleic acids encoding the recombinant, thermostable DNA polymerases, as well as vectors and host cells each including them are also provided. Kits comprising the recombinant, thermostable DNA polymerases are also provided. The recombinant, thermostable DNA polymerases and methods provided herein provide significant cost and efficiency advantages for DNA sequencing.
Gelfand David H.
Kalman Lisa V.
Myers Thomas W.
Reichert Fred L.
Sigua Christopher L.
Borden Ladner Gervais Llp
F. Hoffmann-La Roche Ag
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