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FRET-labeled compounds and uses therefor

  • US 10,066,258 B2
  • Filed: 12/14/2016
  • Issued: 09/04/2018
  • Est. Priority Date: 03/30/2009
  • Status: Active Grant
First Claim
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1. A method of performing nucleic acid sequence analysis, comprising:

  • providing an optical confinement comprising a complex comprising a template nucleic acid and a polymerase enzyme, wherein the complex is capable of template-dependent synthesis of a complementary nascent nucleic acid strand;

    contacting the complex with a reaction mixture to synthesize a nascent nucleic acid strand complementary to the template nucleic acid, wherein the reaction mixture comprises first and second nucleotide analogs, wherein the first nucleotide analog comprises a first fluorescent label that produces a first signal in response to excitation illumination, and the second nucleotide analog comprises a second fluorescent label that produces a second signal in response to excitation illumination, wherein the first and second signals have distinct signal intensities, and wherein the first and second signals comprise peaks at the same wavelengths;

    illuminating the optical confinement with excitation illumination for the first and second fluorescent labels;

    monitoring the illuminated optical confinement to detect signals produced for a plurality of incorporation events in real time during template-dependent synthesis of the nascent nucleic acid strand complementary to the template nucleic acid, wherein the first signal is detectable during incorporation of the first nucleotide analog into the complementary nascent nucleic acid strand and the second signal is detectable during incorporation of the second nucleotide analog into the complementary nascent nucleic acid strand, wherein during incorporation of the first or second nucleotide analogs the first or second fluorescent labels are removed;

    identifying a detected signal as a first signal or a second signal based upon its signal intensity; and

    correlating the identified first or second signal to the incorporation of the first or second nucleotide analog into the nascent nucleic acid strand, respectively.

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