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Recombinant polymerases for incorporation of protein shield nucleotide analogs

  • US 9,873,911 B2
  • Filed: 06/21/2016
  • Issued: 01/23/2018
  • Est. Priority Date: 10/01/2012
  • Status: Active Grant
First Claim
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1. A method of sequencing a DNA template, the method comprising:

  • a) providing a reaction mixture comprising;

    the DNA template,a replication initiating moiety that complexes with or is integral to the template,at least one nucleotide analog, which analog comprises a polypeptide moiety positioned between a dye moiety and a nucleotide component of the analog, wherein the polypeptide moiety comprises at least 60 amino acids; and

    a recombinant DNA polymerase, which recombinant polymerase comprises an amino acid sequence that is at least 90% identical to SEQ ID NO;

    1 or at least 90% identical to SEQ ID NO;

    2, and which recombinant polymerase comprises one or more mutation selected from the group consisting of an amino acid substitution at position Q99, an amino acid substitution at position A134, an amino acid substitution at position K138, an amino acid substitution at position V141, an amino acid substitution at position L142, an amino acid substitution at position A256, an amino acid substitution at position R306, an amino acid substitution at position R308, an amino acid substitution at position K311, an amino acid substitution at position T441, an amino acid substitution at position E466, an amino acid substitution at position D476, an amino acid substitution at position S487, an amino acid substitution at position E508, an amino acid substitution at position L513, an amino acid substitution at position D523, an amino acid substitution at position I524, an amino acid substitution at position K536, an amino acid substitution at position P558, an amino acid substitution at position D570, and an amino acid substitution at position T571, wherein identification of positions is relative to SEQ ID NO;

    1, wherein the recombinant polymerase is capable of replicating at least a portion of the template using the moiety in a template-dependent polymerization reaction;

    b) subjecting the reaction mixture to a polymerization reaction in which the recombinant polymerase replicates at least a portion of the template in a template-dependent manner, whereby the at least one nucleotide analog is incorporated into the resulting DNA; and

    c) identifying a time sequence of incorporation of the at least one nucleotide analog into the resulting DNA.

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