Third Generation DNA Sequencing with a Nanopore - Nanopores: Sensing and Fundamental Biological Interactions - page 305

histograms of the current fluctuations like that shown in Fig. 12.7e , which can be
represented by the superposition of two Gaussian distributions: one (solid blue)
offset from the median ( DI ¼ 0) by DI ¼ +2.9 pA with a width of s ¼ 7.8 pA; and

another (solid red) offset by DI ¼ 6.5 pA with a width of s ¼ 5.9 pA. The

2 -statistic, improves from 2

w ¼

goodness of fit, measured by the reduced w 5.04

2

for a single Gaussian fit to w ¼

0.71 for two Gaussians, indicating that two
Gaussians represent a superior model. This is in contrast to the open pore current
data shown in Fig. 12.7f , which can be fit by a single Gaussian with a width

s ¼ 4.3

2

pA with w 0.67 measured at the same voltage.

We tentatively attribute the separate peaks in Fig. 12.7e at DI ¼ +2.9 pA and
DI ¼

¼

6.5 pA to partially resolved signals associated with C-G/G-C and A-T/T-A
base-pairs, respectively. This identification is supported by the observation that
l

-DNA has a nearly uniform distribution of base-pairs T-A, A-T, C-G, G-C. Thus,
the currents corresponding to each type of base-pair should translate to approxi-
mately equal, distinct distributions in the blockade current convolved with the
electrical noise. The difference between C-G and A-T base-pairs can be resolved
in this case because of the long (~2 ms) time each base-pair spends in the
constriction. However, apparently the SNR is inadequate for discriminating C-G
from G-C or A-T from T-A, despite the slowing. This assertion is also corroborated
by even longer duration measurements of blockade currents associated with strep-
tavidin bound, 100 bp long, C-G and A-T biotinylated duplexes trapped by the
electric field in a pore in a configuration described elsewhere [ 65 ].
One obvious problem with sequencing this way is determining which nucleotide
is on which strand. However, our simulations show that the base-pair tilt, caused by
the confinement, is maintained during a translocation with the nucleotides of one
strand always lagging their partners on the other. At low bias, the electric potential
of the nucleotides in the tilted configuration presents a peculiar energy barrier to the
ions with a passage rate that is exponentially related to the height. The differences
in the heights for different sequences could therefore have substantial effects on the
current-voltage relation.

12.2.4 Noise in a Nanopore

Thus, the current measurement shown in Fig. 12.7c apparently corresponds to a
single long read of 10,000 bp! While trapping a DNA molecule facilitates the

high fidelity reads required for sequencing it, the electrical noise associated with the
pore current introduces ambiguity that adversely affects base-calling. Both the high
frequency and noise performance of the pore current are critical for applications like
sequencing. High throughput demands a high translocation velocity of the DNA, and
while a solid-state nanopore can satisfy this specification, the noise is proportional to
the bandwidth so that the fidelity of base-calling may be compromised.
According to the model of Smeets et al. [ 66 ], the frequency response is essen-
tially determined by the series combination of of the membrane capacitance, C m ,

>