Nanopore-based polymer analysis with mutually-quenching fluorescent labels
First Claim
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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Abstract
The invention is directed to a method for determining a monomer sequence of a polymer that is translocated through a nanopore. Monomers of the polymer are labeled with fluorescent labels such that in free solution fluorescent labels of adjacent monomers substantially quench each other and wherein the nanopore constrains fluorescent labels within its bore into a constrained state wherein no detectable fluorescent signal can be generated. By exciting the fluorescent label of each monomer as it exits the nanopore and transitions from a constrained state to a quenched state with an adjacent fluorescent label, a fluorescent signal can be generated by the exiting fluorescent label that allows its monomer to be identified, thereby permitting a monomer sequence to be determined from a sequence of fluorescent signals as the polymer translocates through the nanopore.
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Citations
20 Claims
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1. A method of determining a nucleotide sequence of at least one polynucleotide, the method comprising the steps of:
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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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 17, 18, 19, 20)
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11. A method of determining a nucleotide sequence of at least one polynucleotide, the method comprising the steps of:
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translocating at least one single stranded polynucleotide through a nanopore, wherein substantially all different kinds of nucleotides of the single stranded polynucleotide are labeled with adducts and/or 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 so that substantially no detectable signal is generated and wherein the adducts and fluorescent labels are selected so that translocation speed through the nanopore is reduced to permit detection of individual nucleotides and wherein each adduct is an organic molecule having a molecular weight in the range of that of an organic dye and wherein each fluorescent label of the mutually quenching set (i) quenches fluorescence of every other fluorescent label of the same set whenever such labels are attached to adjacent nucleotides of a polynucleotide in free solution, and (ii) generates a distinct fluorescent signal in a non-quenched stat; 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. - View Dependent Claims (12, 13, 14, 15, 16)
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Specification