NANOPORE-BASED NUCLEIC ACID ANALYSIS WITH MIXED FRET DETECTION
Various methods, systems and devices for optical detection and analysis of polymers, such as polynucleotides, using nanopores, e.g., for determining sequences of nucleic acids, are provided herein. In certain variations, methods and systems for determining a nucleotide sequence of a polynucleotide, which include measuring mixed FRET signals as a polynucleotide translocates through a nanopore and determining a nucleotide sequence of the polynucleotide from the mixed FRET signals, are provided.
1 .- 15 . (canceled)
16 . A system for determining a nucleotide sequence of a polynucleotide, the system comprising:
a nanopore providing fluid communication between a first chamber and a second chamber and through which a polynucleotide can be translocated, the nanopore being dimensioned so that nucleotides of the polynucleotide pass through an exit of the nanopore in sequence and whenever nucleotides of the polynucleotide are labeled with FRET acceptors, FRET is suppressed between such FRET acceptors inside the nanopore and a FRET donor outside the nanopore;
a polynucleotide with acceptor-labeled nucleotides wherein different kinds of nucleotides are labeled with acceptors that generate distinguishable signals;
a FRET donor disposed within a FRET distance of the exit of the nanopore, such that a plurality of nucleotides pass within a FRET distance of the FRET donor upon emerging from the exit of the nanopore so that a mixed FRET signal is generated;
a light source for exciting the FRET donor; and
a detector for collecting mixed FRET signals and separating distinguishable signals from two or more different acceptors.
17 . The system of claim 16 , wherein said nanopore is disposed in a solid phase membrane and wherein said FRET donor is attached to the solid phase membrane adjacent to said nanopore.
18 . The system of claim 16 , wherein said nanopore is a protein nanopore and wherein said FRET donor is attached to the protein nanopore.
19 . The system of claim 16 , wherein the polynucleotide is a single stranded polynucleotide.
20 . (canceled)
21 . The system of claim 16 , wherein at least one of said acceptors is a fluorescent organic dye.
22 . The system of claim 16 , wherein said FRET donor is a quantum dot.
23 . The system of claim 16 , wherein said step of exciting said FRET donor includes exposing said FRET donor to an illumination beam.
24 - 26 . (canceled)
27 . The system of claim 16 , wherein said nucleotide sequence of said polynucleotide is determined from said separated distinguishable signals collected from every nucleotide of said polynucleotide.