Sequence-specific detection of individual DNA strands using engineered nanopores.
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 alpha-hemolysin (alphaHL) 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.
Howorka
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S
Department of Medical Biochemistry and Genetics, The Texas A&M University System Health Science Center, 440 Reynolds Medical Building, College Station, TX 77843-1114, USA. howorka@medicine.tamu.edu
Cheley
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S
Bayley
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H
D020137:Base Pair Mismatch; D015374:Biosensing Techniques; D001709:Biotechnology / Q000379:methods; D002462:Cell Membrane / Q000737:chemistry; D004247:DNA / Q000032:analysis* / Q000378:metabolism; D006678:HIV / Q000201:enzymology; D008051:Lipid Bilayers / Q000378:metabolism; D008954:Models, Biological; D009154:Mutation; D019829:Nevirapine / Q000494:pharmacology; D009693:Nucleic Acid Hybridization / Q000379:methods*; D012194:RNA-Directed DNA Polymerase / Q000235:genetics; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology; D017422:Sequence Analysis, DNA / Q000379:methods*; D013997:Time Factors
D008051:Lipid Bilayers; D018894:Reverse Transcriptase Inhibitors; D004247:DNA; D019829:Nevirapine; D012194:RNA-Directed DNA Polymerase