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Reagents, methods, and libraries for bead-based sequencing

  • US 9,493,830 B2
  • Filed: 10/18/2013
  • Issued: 11/15/2016
  • Est. Priority Date: 02/01/2005
  • Status: Active Grant
First Claim
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1. A method for determining a sequence of a nucleic acid template, comprising:

  • (I) interrogating the nucleic acid template with a plurality of probes from at least two distinguishably labeled probe families, wherein each probe of the plurality of probes comprises a scissile linkage between a constrained portion of the probe and a distal portion of the probe that is not extendable by a ligase, and wherein the distal portion of the probe further comprises a label, wherein interrogating comprises;

    (a) forming a duplex by hybridizing a first initializing oligonucleotide to the template at a primer binding sequence;

    (b) extending the first initializing oligonucleotide hybridized to the template by ligating a labeled probe from the plurality of probes that has hybridized to the template to the initializing oligonucleotide;

    (c) detecting a signal corresponding to the label of the ligated probe to identify its probe family;

    (d) cleaving the probe at the scissile linkage and removing the label;

    (e) repeating successive cycles of steps (b) to (d) forming an extension strand;

    (f) generating a first ordered series of signals corresponding to a first region of the template;

    (g) removing the extension strand; and

    (h) repeating steps (a) to (g) with a second initializing oligonucleotide and generating a second ordered series of signals corresponding to a second region of the template, the second region differing from the first region and overlapping the first region by at least one nucleic acid;

    (II) recording the ordered series of signals, corresponding to the first and second region of the template, on a computer readable medium;

    (III) obtaining sequence information for the first region of the template from the first series of signals and sequence information for the second region of the template from the second series of signals; and

    (IV) determining a sequence of the nucleic acid template based in part on the sequence information from the first and second regions.

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