Nanopore Analysis Using Sequencespecific Detection

Howorka et al. [23] found that it was possible to attach a single-stranded oligomer of DNA to hemolysin so that the vestibule contained a short sequence that potentially could bind to traversing DNA if complementary base pairing was possible. This remarkable feat of nanoengineering produced a biosensor that could detect specific DNA sequences with single base resolution. The principle underlying the function of this biosensor is that a traversing DNA strand binds to the tethered DNA in the vestibule by complementary base pairing and remains in the pore for a measurable amount of time. As a result, a distinctive two-part signal is generated, first by the duplex DNA that partially blocks the current, followed by a spike as the target DNA is released and passes through the pore stem. DNA that lacks the complementary base pairing passes through very rapidly without the partial blockade. The duplex lifetimes allow discrimination between short DNA strands that differ by only a single nucleotide so that duplex formation is inhibited. Because of this sensitivity, it was possible to determine the sequence of a codon embedded in the sequence of a single DNA oligomer cova-lently linked to the vestibule. These authors also used this approach to detect a mutation in the reverse transcriptase gene of the HIV virus.

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