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DNA stretching and optimization of nucleobase recognition in enzymatic nanopore sequencing

Stoddart, D., Franceschini, L., Heron, A., Bayley, H. & Maglia, G., 27-Feb-2015, In : Nanotechnology. 26, 8, 6 p., 084002.

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  • DNA stretching and optimization of nucleobase recognition in enzymatic nanopore sequencing

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DOI

In nanopore sequencing, where single DNA strands are electrophoretically translocated through a nanopore and the resulting ionic signal is used to identify the four DNA bases, an enzyme has been used to ratchet the nucleic acid stepwise through the pore at a controlled speed. In this work, we investigated the ability of alpha-hemolysin nanopores to distinguish the four DNA bases under conditions that are compatible with the activity of DNA-handling enzymes. Our findings suggest that in immobilized strands, the applied potential exerts a force on DNA (similar to 10 pN at +160 mV) that increases the distance between nucleobases by about 2.2 angstrom V-1. The four nucleobases can be resolved over wide ranges of applied potentials (from +60 to +220 mV in 1 m KC1) and ionic strengths (from 200 mM KC1 to 1M KC1 at +160 mV) and nucleobase recognition can be improved when the ionic strength on the side of the DNA-handling enzyme is low, while the ionic strength on the opposite side is high.

Original languageEnglish
Article number084002
Number of pages6
JournalNanotechnology
Volume26
Issue number8
Publication statusPublished - 27-Feb-2015

    Keywords

  • DNA stretching, electroosmosis, single-molecule, ionic strength, SINGLE-STRANDED-DNA, ALPHA-HEMOLYSIN, MEMBRANE CHANNEL, NUCLEOTIDE RESOLUTION, BIOLOGICAL NANOPORE, TRANSMEMBRANE PORE, PROTEIN NANOPORE, TRANSLOCATION, POLYMERASE, MOLECULES

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