Sequence-specific detection of individual DNA strands using engineered nanopores

We describe biosensor elements that are capable of identifying individual D NA strands with single-base resolution. Each biosensor element consists of an individual DNA oligonucleotide covalently attached within the lumen of t he cc-hemolysin (alpha HL) pore to form a "DNA-nanopore". The binding of si ngle-stranded DNA (ssDNA) molecules to the tethered DNA strand causes chang es in the ionic current flowing through a nanopore. On the basis of DNA dup lex lifetimes. the DNA-nanopores are able to discriminate between individua l DNA strands up to 30 nucleotides in length differing by a single base sub stitution. This was exemplified by the detection of a drug resistance-confe rring mutation in the reverse transcriptase gene of HIV. In addition, the a pproach was used to sequence a complete codon in an individual DNA strand t ethered to a nanopore.