Approaches to nanopore sequencing. (a) Strand-sequencing using ionic current blockage. A typical trace of the ionic current amplitude (left) through an α-hemolysin pore clearly differentiates between an open pore (top right) and one blocked by a strand of DNA (bottom right) but cannot distinguish between the ~12 nucleotides that simultaneously block the narrow transmembrane channel domain (red bracket). (b) Exonuclease-sequencing by modulation of the ionic current. An exonuclease (pale blue) attached to the top of an α-hemolysin pore through a genetically encoded (deep blue), or chemical, linker sequentially cleaves dNMPs (gold) off the end of a DNA strand (in this case, one strand of a double-stranded DNA). A dNMP's identity (A, T, G or C) is determined by the level of the current blockade it causes when driven into an aminocyclodextrin adaptor (red) lodged within the pore. After a few milliseconds, the dNMP is released and exits on the opposite side of the bilayer. (c) Nanopore sequencing using synthetic DNA and optical readout. Each nucleotide in the target DNA that is to be sequenced is first converted into a longer DNA strand composed of pairs of two different code-units (colored orange and blue for illustration); each code-unit is a 12-base-long oligomer. After hybridizing the converted DNA with molecular beacons that are complementary to the code units, these beacons are stripped off using a nanopore. The sequence of the original DNA is read by detecting the discrete short-lived photon-bursts as each oligo is stripped. (d) Strand-sequencing using transverse electron currents. DNA is driven through a nanopore functionalized with embedded emitter and collector tunneling probes (orange) and a back gate (black). The amplitude of the tunneling currents that traverse through the nucleotides is expected to differentiate each nucleobase as the DNA is electrophoretically driven through the pore (arrow).

A nanopore reader with chemically functionalized probes. As a strand of DNA emerges from a nanopore, a `phosphate grabber' on one functionalized electrode and a `base reader' on the other electrode form hydrogen bonds (light blue ovals) to complete a transverse electrical circuit through each nucleotide as it is translocated through the nanopore.