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Sequence-specific detection of individual DNA strands using engineered nanopores

Abstract

We describe biosensor elements that are capable of identifying individual DNA strands with single-base resolution. Each biosensor element consists of an individual DNA oligonucleotide covalently attached within the lumen of the α-hemolysin (αHL) pore to form a “DNA–nanopore”. The binding of single-stranded DNA (ssDNA) molecules to the tethered DNA strand causes changes in the ionic current flowing through a nanopore. On the basis of DNA duplex lifetimes, the DNA–nanopores are able to discriminate between individual DNA strands up to 30 nucleotides in length differing by a single base substitution. This was exemplified by the detection of a drug resistance–conferring mutation in the reverse transcriptase gene of HIV. In addition, the approach was used to sequence a complete codon in an individual DNA strand tethered to a nanopore.

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Figure 1: A single DNA oligonucleotide attached to the αHL pore.
Figure 2: A single-base mismatch abolishes the binding of an oligonucleotide to a tethered DNA strand within a DNA–nanopore.
Figure 3: A DNA–nanopore detects a common mutation, which confers resistance to the drug nevirapine in the reverse transcriptase gene of HIV.

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Acknowledgements

The authors thank Stephen Cheley for providing the plasmid construct αHL-WT-RL-D4 and Orit Braha and Liviu Movileanu for helpful discussions. This work was supported by the US Department of Energy, NIH, the Office of Naval Research (Multidisciplinary University Research Initiative 1999), and the Texas Advanced Technology Program. S.H. is currently recipient of a fellowship from the Max-Kade Foundation and was supported by a postdoctoral scholarship from the Austrian Science Foundation (Fonds zur Förderung der wissenschaftlichen Forschung) during the earlier stages of the work.

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Correspondence to Stefan Howorka.

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Howorka, S., Cheley, S. & Bayley, H. Sequence-specific detection of individual DNA strands using engineered nanopores. Nat Biotechnol 19, 636–639 (2001). https://doi.org/10.1038/90236

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