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 .- 26 . (canceled) 
     
     
         27 . A method of identifying a polymer, the method comprising the steps of:
 translocating a polymer through a nanopore, wherein different kinds of monomers of the polymer 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 set whenever such labels are attached to adjacent monomers of a polymer in free solution, and (ii) generates a distinct fluorescent signal when excited and when in a non-quenched state;   exciting the fluorescent label of each labeled monomer upon exiting the nanopore and during its transition from a constrained state to formation of a quenched state with an adjacent monomer;   measuring a fluorescent signal generated by the exiting fluorescent label to identify the monomer to which the fluorescent label is attached; and   identifying a polymer from a sequence of fluorescent signals.   
     
     
         28 . The method of  claim 27  wherein said polymer is a polypeptide and said monomers are amino acids. 
     
     
         29 . The method of  claim 28  wherein at least two different amino acids are labeled with different fluorescent labels. 
     
     
         30 . The method of  claim 28  wherein said mutually quenching set contains consists of two fluorescent dyes. 
     
     
         31 . The method of  claim 27  wherein said nanopore is disposed in a lipid bilayer on a solid phase membrane. 
     
     
         32 . The method of  claim 31  wherein said nanopore is a protein nanopore. 
     
     
         33 . The method of  claim 32  wherein said protein nanopore is a hemolysin or a variant thereof. 
     
     
         34 . The method of  claim 27  wherein said step of exciting includes exposing said exiting fluorescent label to an evanescent wave from a total internally reflected excitation. 
     
     
         35 . The method of  claim 27  wherein said step of exciting includes exciting by epifluorescent microscopy. 
     
     
         36 . The method of  claim 27  wherein said nanopore is in a nanopore array comprising a plurality of substantially identical nanopores. 
     
     
         37 . The method of  claim 36  wherein said nanopore array comprises a solid phase membrane separating a first chamber from a second chamber, the solid phase membrane comprising a planar array of apertures each having said protein nanopore immobilized therein. 
     
     
         38 . The method of  claim 27  wherein one or more of said fluorescent labels are selected from among rhodamine dyes, fluorescein dyes and cyanine dyes.