Nanopore-based nucleic acid analysis with mixed fret detection

Abstract

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 . A method of determining a nucleotide sequence of a polynucleotide, the method comprising the steps of:
 translocating a polynucleotide through a nanopore so that nucleotides of the polynucleotide pass in sequence by a first member of a FRET pair positioned adjacent to the nanopore, a plurality of the nucleotides being within a FRET distance of the first member of the FRET pair as the nucleotides exit the nanopore and at least a portion of the nucleotides being labeled with a second member of the FRET pair;   exposing the FRET pairs adjacent to the nanopore to a light beam so that FRET occurs between the first and a plurality of second members of the FRET pair within the FRET distance to generate a mixed FRET signal;   measuring mixed FRET signals as the polynucleotide translocates through the nanopore; and   determining a nucleotide sequence of the polynucleotide from the mixed FRET signals.   
     
     
         2 . The method of  claim 1 , wherein said nanopore is disposed in a solid phase membrane and wherein said first member of said FRET pair is attached to the solid phase membrane adjacent to said nanopore. 
     
     
         3 . The method of  claim 1 , wherein said nanopore is a protein nanopore and wherein said first member of said FRET pair is attached to the protein nanopore. 
     
     
         4 . The method of  claim 1 , wherein the polynucleotide is a single stranded or double stranded polynucleotide. 
     
     
         5 - 8 . (canceled) 
     
     
         9 . A method of determining a nucleotide sequence of a polynucleotide, the method comprising the steps of:
 translocating a polynucleotide with labeled nucleotides through a nanopore dimensioned so that labels on the nucleotides are constrained to suppress FRET reactions, the labels on the nucleotides being second members of a FRET pair, and so that nucleotides of the polynucleotide pass in sequence through a FRET zone upon exiting the nanopore, the FRET zone encompassing a plurality of the nucleotides during such passage and at least one first member of the FRET pair being in the FRET zone;   exposing the first and second members of the FRET pair in the FRET zone to a light beam so that FRET occurs between the first and second members to generate a mixed FRET signal;   measuring mixed FRET signals as the polynucleotide moves through the FRET zone; and   determining a nucleotide sequence of the polynucleotide from the mixed FRET signals.   
     
     
         10 . The method of  claim 9 , wherein said nanopore is disposed in a solid phase membrane and wherein said first member of said FRET pair is attached to the solid phase membrane adjacent to said nanopore. 
     
     
         11 . The method of  claim 9 , wherein said nanopore is a protein nanopore and wherein said first member of said FRET pair is attached to the protein nanopore. 
     
     
         12 . The method of  claim 9 , wherein the polynucleotide is a single stranded or double stranded polynucleotide. 
     
     
         13 . The method of  claim 9 , wherein said first members of said FRET pair are donors and said second members of said FRET pair are acceptors. 
     
     
         14 . The method of  claim 13 , wherein at least one of said acceptors is a fluorescent organic dye. 
     
     
         15 . The method of  claim 13 , wherein at least one of said donors is a quantum dot. 
     
     
         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 a member of a FRET pair, FRET is suppressed between such members inside the nanopore and those outside the nanopore; and   a member of the FRET pair disposed within a FRET distance of the exit of the nanopore, wherein the member of the FRET pair disposed within a FRET distance of the exit of the nanopore is a different member than a member labeling the polynucleotide, such that a plurality of nucleotides pass within a FRET distance of the disposed member upon emerging from the exit of the nanopore.   
     
     
         17 . The system of  claim 16 , wherein said nanopore is disposed in a solid phase membrane and wherein said first member of said FRET pair 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 first member of said FRET pair is attached to the protein nanopore. 
     
     
         19 . The system of  claim 16 , wherein the polynucleotide is a single stranded or double stranded polynucleotide. 
     
     
         20 . The system of  claim 16 , wherein the disposed member of said FRET pair is a donor and the member labeling the polynucleotide is an acceptor. 
     
     
         21 . The system of  claim 20 , wherein the acceptor is a fluorescent organic dye. 
     
     
         22 . The system of  claim 20 , wherein the donor is a quantum dot.