Nanopore-Based DNA Sequencing and DNA Motion Control - Nanopores: Sensing and Fundamental Biological Interactions - page 271

11.1.4.6 Oxford Nanopore DNA Sequencing

The Oxford nanopore DNA sequencing approach [ 28 ] employs an exonuclease
enzyme to cleave individual nucleotide molecules from the target DNA and drives
them through a protein nanopore, which is a hemolysin mutant illustrated as
WT-(M113R/N139Q)6(M113R/N139Q/L135C)1. The protein nanopore is cova-
lently attached to an adaptor molecule, heptakis(6-deoxy-6-amino)-6-N-mono
(2-pyridyl)dithiopropanoyl- CD), which will slow

down the motion of nucleotide molecules through the pore [ 28 ]. By cleaving
the target DNA molecule into individual nucleotide, one does not need to worry
about the secondary structure of DNA or the length of the nanopore channel
(previously mentioned conditions (1) and (2)). By putting an adaptor molecule

b -cyclodextrin (am 6 amPDP 1 b

Fig. 11.19 Nucleotide event distributions with the adapter. (a) Single-channel recording from the
WT-(M113R/N139Q) 6 (M113R/N139Q/L135C) 1 -am6amDP 1 CD pore showing dGMP, dTMP,

dAMP and dCMP discrimination, with shaded bands (three standard deviations from the centre
of the individual Gaussian fits) added to represent the residual current distribution for each
nucleotide. (b) Corresponding residual current histogram of nucleotide binding events, including
Gaussian fits. Data acquired in 400 mM KCl, 25 mM Tris HCl, pH 7.5, at 180 mV in the presence
of 10 mM dGMP, 10 mM dTMP, 10 mM dAMP and 10 mM dCMP. Adapted from [ 28 ]

b