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POLYMERASES ENGINEERED TO REDUCE NUCLEOTIDE-INDEPENDENT DNA BINDING

  • US 20200131486A1
  • Filed: 01/09/2020
  • Published: 04/30/2020
  • Est. Priority Date: 04/29/2016
  • Status: Active Application
First Claim
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1. A method of detecting a next correct nucleotide of a primed template nucleic acid, comprising the steps of:

  • (a) contacting the primed template nucleic acid with a first reaction mixture that comprises a crippled DNA polymerase and a test nucleotide,wherein the test nucleotide is the next correct nucleotide of the primed template nucleic acid,wherein a ternary complex is formed, the ternary complex comprising the primed template nucleic acid, the crippled DNA polymerase and the test nucleotide, andwherein the crippled DNA polymerase comprises a variant of the sequence of SEQ ID NO;

    3, said variant being at least 80% identical to SEQ ID NO;

    3 and comprising an amino acid substitution mutation at one or more of positions K250, Q281, D355, Q425, and D532; and

    (b) detecting the ternary complex without chemical incorporation of the test nucleotide into the primer of the primed template nucleic acid, thereby detecting the next correct nucleotide.

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