Nanopore-Based DNA Sequencing and DNA Motion Control - Nanopores: Sensing and Fundamental Biological Interactions - page 272

inside the protein nanopore, one also solves the concerns of previously mentioned
condition (3) and (4): characteristic transient current signatures for each nucleotide
molecules and enough dwelling time of each nucleotide molecule inside the
nanopore.
Figure 11.19a shows the ionic current trace of 10 mM dGMP, 10 mM dTMP,
10 mM dAMP and 10 mM dCMP in 400 mM KCl, 25 mM Tris HCl, pH 7.5, at
180 mV voltage bias across the hemolysin mutant pore with the adaptor molecule.
Individual nucleotide molecules dGMP, dTMP, dAMP and dCMP can be discri-
minated based on the reduced ionic current while they bind in the nanopore [ 28 ], as
illustrated by the shaded bands. Residual current histogram of nucleotide binding
events, including Gaussian fits are shown in Fig. 11.19b , with clear separation of
Gaussian peaks for each type of nucleotide molecules [ 28 ]. Demonstration of
differentiating individual nucleotide molecules is a significant achievement and
the remaining challenge of this approach is to guarantee that the cleaved nucleotide
molecules will be transported to the pore in the original sequence of the target
DNA molecule.
11.2 DNA Motion Control in the Nanopore
Among the various nanopore DNA sequencing approaches, electrical sensor
approaches [ 19 , 20 , 24 ] require no modifications on the target single stranded
DNA molecules, and thus have the potential to attain the lowest cost. For electrical
sensor approaches, simulations [ 19 , 21 , 24 ] have suggested that the detection of
different electrical signals for different DNA bases is plausible assuming that the
position of DNA is well controlled against thermal agitation. Whatever electrical
sensor is proposed for differentiating individual DNA bases while DNA is moved
through a nanopore, the motion of the DNA has to be controlled at single base
resolution. Otherwise the readings from the sensor will not be able to tell the
“sequence” of the bases. Thus, controlling the motion of DNA translocation
through a nanopore is a key challenge in the nanopore DNA sequencing field,
and is the topic of this section.
11.2.1 Viscosity, Voltage, Ionic Concentration
and Temperature Control
As demonstrated by Fologea et al . [ 31 ], the translocation speed of DNA through the
nanopore is affected by the viscosity of the buffer (Fig. 11.20 ), the applied voltage
bias (Fig. 11.21 ), the salt concentration (Fig. 11.22 ) and the temperature of the
buffer (Fig. 11.23 ). DNA translocation speed can be considerably slowed down by
increasing viscosity, decreasing applied voltage bias, decreasing the ionic