THERMOSTABLE DNA POLYMERASES INCORPORATING NUCLEOSIDE TRIPHOSPHATES LABELED WITH FLUORESCEIN FAMILY DYES

摘要

A recombinant, thermostable DNA polymerase is provided herein. Such polymerasecomprises the amino acid sequence LeuSerXaaXaaLeuXaaXaaProXaaXaaGlu (SEQ IDNO:1), wherein "Xaa" at positions 3, 6, 9, and 10 of the sequence are any aminoacid residue,"Xaa" at position 4 in the sequence is a polar amino acid, or a positively-charged amino acidand "Xaa" at position 7 of the sequence is Val or Ile. The recombinant,thermostable DNApolymerase has a level of discrimination against incorporation of nucleotideswhich arelabeled with fluorescein family dyes which is reduced in comparison to thenative form of thepolymerase. Such modified recombinant, thermostable DNA polymerases haveenhancedefficiency for incorporating unconventional nucleotides, e.g., those labeledwith fluoresceinfamily dyes. Such modified recombinant, thermostable DNA polymerases can beprepared bymethods of recombinant DNA technology. Such recombinant, thermostable DNApolymerases are advantageous in many in vitro DNA synthesis applications,e.g., DNAsequencing, synthesis of labeled DNA or the production of labeled primerextension products.Such recombinant, thermostable DNA polymerases are particularly useful inchaintermination nucleic acid sequencing methods. Nucleic acids encoding therecombinant,thermostable DNA polymerases, as well as vectors and host cells each includingthem arealso provided. Kits comprising the recombinant, thermostable DNA polymerasesare alsoprovided. The recombinant, thermostable DNA polymerases and methods providedhereinprovide significant cost and efficiency advantages for DNA sequencing.