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Pyrosequencing methods and related compositions

  • US 9,169,510 B2
  • Filed: 06/20/2006
  • Issued: 10/27/2015
  • Est. Priority Date: 06/21/2005
  • Status: Active Grant
First Claim
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1. A method for determining the nucleotide sequence of a single-stranded DNA comprising performing the following steps:

  • (a) immobilizing the single-stranded DNA on a solid substrate via an azido linkage, an alkynyl linkage, or a 1,2,3-triazole linkage;

    (b) contacting the DNA under DNA polymerization-permitting conditions with (i) a 3′

    -O-blocked dNTP selected from the group consisting of 3′

    -O-blocked dATP, 3′

    -O-blocked dCTP, 3′

    -O-blocked dGTP, and 3′

    -O-blocked dTTP, wherein each 3′

    -O-blocked dNTP comprises an unmodified triphosphate group, and wherein each 3′

    -O-blocked dNTP comprises an unlabeled base, and (ii) 9°

    N DNA polymerase (exo-) A4851/Y409V, wherein the moiety blocking the 3′

    -O atom of the dNTP is an allyl moiety or a methoxymethyl moiety;

    (c) (i) determining whether pyrophosphate is generated as a result of step (b), whereby (1) pyrophosphate generation indicates that polymerization has occurred and the identity of the nucleic acid residue in the DNA is that which is complementary to the 3′

    -O-blocked dNTP used in part (i) of step (b), and (2) the absence of pyrophosphate generation indicates that the identity of such nucleic acid residue is not that which is complementary to such 3′

    -O-blocked dNTP;

    wherein determining whether pyrophosphate is generated is performed by detecting dissociation of a coumarin-derived indicator from a complex between the indicator and a bis-Zn2+-dipicolylamine coordination compound, wherein the coumarin-derived indicator has the following structure;

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