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Modified polymerases for improved incorporation of nucleotide analogues

  • US 10,696,955 B2
  • Filed: 11/28/2018
  • Issued: 06/30/2020
  • Est. Priority Date: 09/30/2014
  • Status: Active Grant
First Claim
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1. A method for incorporating modified nucleotides into DNA comprising allowing the following components to interact:

  • (i) a DNA template;

    (ii) a nucleotide solution; and

    (iii) a recombinant DNA polymerase comprising an amino acid sequence that is at least 80% identical to SEQ ID NO;

    10, which recombinant DNA polymerase comprises at least one amino acid substitution mutation at one or more positions functionally equivalent to Thr144, Gly153, Lys476, Leu478, Thr590, Ala639 or Asp718 in the 9°

    N DNA polymerase amino acid sequence, wherein the mutation at the position functionally equivalent to Gly153 comprises a mutation to a polar amino acid, wherein the mutation at the position functionally equivalent to Lys476 comprises a mutation to a hydrophobic amino acid, wherein the mutation at the position functionally equivalent to Leu478 comprises a mutation to a polar amino acid, wherein the mutation at the position functionally equivalent to Asp718 comprises a mutation homologous to Asp718Asn, and wherein the DNA polymerase is a family B DNA polymerase.

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