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Nanopore-based polymer analysis with mutually-quenching fluorescent labels

  • US 9,885,079 B2
  • Filed: 10/08/2015
  • Issued: 02/06/2018
  • Est. Priority Date: 10/10/2014
  • Status: Active Grant
First Claim
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1. A method of determining a nucleotide sequence of at least one polynucleotide, the method comprising the steps of:

  • translocating at least one single stranded polynucleotide through a nanopore, wherein different kinds of nucleotides of the single stranded polynucleotide are labeled with different fluorescent labels from a mutually quenching set and wherein the nanopore has a bore dimensioned to force the fluorescent labels within the nanopore into a constrained state wherein substantially no detectable signal is generated and wherein each fluorescent label of the mutually quenching set (i) quenches fluorescence of every other fluorescent label of the same set whenever inch labels are attached to adjacent nucleotides of a polynucleotide in free solution, and (ii) generates a distinct fluorescent signal in a non-quenched state;

    exciting the fluorescent label of each nucleotide upon exiting the nanopore and during its transition from a constrained state to formation of a quenched state with an adjacent nucleotide;

    measuring a fluorescent signal generated by the exiting fluorescent label to identify the nucleotide to which the fluorescent label is attached; and

    determining a nucleotide sequence of the polynucleotide from a sequence of fluorescent signals.

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