Abstract: The invention provides methods for analyzing polynucleotides using nanopores that allow passage of single stranded polynucleotides but not double stranded polynucleotides. In accordance with some embodiments, a double-stranded product is produced that comprises a labeled strand with a single stranded tail or overhang. The double stranded product is exposed to one or more nanopores in the presence of an electric field across the one or more nanopores such that the single stranded tail may be captured and the labeled strand translocated by unzipping from the double stranded product. The ionic composition of the reaction mixture and electric field strength are selected so that nucleotides translocate a nanopore at a rate of less than 1000 nucleotides per second.
Abstract: The invention is directed to a device comprising a protein nanopore immobilized in a lipid layer within an aperture of a solid phase substrate, which provides a stable platform for using first and second members of one or more FRET pairs to generate optical signals as a labeled analyte translocates through the bore of the protein nanopore. In another aspect, the invention is directed to the use of the device to determine the nucleotide sequence of a polynucleotide analyte.
Abstract: Methods and systems for sequencing a biological molecule or polymer, e.g., a nucleic acid, are provided. One or more donor labels, which are attached to a pore or nanopore, may be illuminated or otherwise excited. A polymer having a monomer labeled with one or more acceptor labels, may be translocated through the pore. Either before, after or while the labeled monomer of the polymer passes through, exits or enters the pore, energy may be transferred from the excited donor label to the acceptor label of the monomer. As a result of the energy transfer, the acceptor label emits energy, and the emitted energy is detected in order to identify the labeled monomer of the translocated polymer and to thereby sequence the polymer.
Abstract: The invention is directed to a device comprising a protein nanopore immobilized in a lipid layer within an aperture of a solid phase substrate, which provides a stable platform for using first and second members of one or more FRET pairs to generate optical signals as a labeled analyte translocates through the bore of the protein nanopore. In another aspect, the invention is directed to the use of the device to determine the nucleotide sequence of a polynucleotide analyte.
Abstract: Methods and systems for sequencing a biological molecule or polymer, e.g., a nucleic acid, are provided. One or more donor labels, which are attached to a pore or nanopore, may be illuminated or otherwise excited. A polymer having a monomer labeled with one or more acceptor labels, may be translocated through the pore. Either before, after or while the labeled monomer of the polymer passes through, exits or enters the pore, energy may be transferred from the excited donor label to the acceptor label of the monomer. As a result of the energy transfer, the acceptor label emits energy, and the emitted energy is detected in order to identify the labeled monomer of the translocated polymer and to thereby sequence the polymer.