Biomedical Engineering Reference
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Fig. 11.4 Pyrosequencing performed on a 25-base-long oligonucleotide template. Proportional
signals are obtained for one, two, three and four base incorporations. Repeated cycles of A, G, T, C
incorporations are indicated at the bottom part of the figure, while the top part of the figure
illustrates the obtained sequence based on the intensity of the fluorescence signal. Adapted from [
8
]
11.1.4 Nanopore-Based DNA Sequencing: Challenges
and Opportunities
11.1.4.1 KBBD Proposal
Nanopore-based DNA sequencing is a non-cyclical, single-molecule, real-time DNA
sequencing method. It was first proposed in 1996 by Kasianowicz et al
.
in their
seminal paper [
11
] on DNA translocation. A biological protein channel (alpha-
hemolysin, or alpha-HL, with 1.5 nm pore diameter) can form in a diphytanoyl
phosphatidylcholine lipid bilayer (Fig.
11.5
), which is across a horizontal, conical
aperture at the end of a Teflon tube (Fig.
11.6
). The ionic current through the nanopore
can be detected using the Patch-Clamp electronics when a voltage is applied across
the membrane. Due to the volume exclusion, passage of molecules will decrease the
number of ions inside the nanopore, thus decreasing the ionic current. As shown in
Fig.
11.7
, the passage of single-stranded DNA molecules through this membrane-
embedded
-hemolysin channel, for example, causes a ~90% blockage of the ionic
current at 1 M KCl solution. This fact led to the KBBD proposal (for Kasianowicz,
Brandin, Branton and Deamer), that it may be possible to use the ionic current passing
through the nanopore to directly determine the sequence of a polynucleotide.
One obvious experiment is to check the current blockades caused by homopo-
lymers of polycytidylic acid (poly (C)), polyadenylic acid (poly (A)). A single
adenylic base is larger than a single cytidylic base, as shown in Fig.
11.8
, one would
expect that the current blockage of poly (A) is larger than that of poly (C). But the
experiment done by Akeson et al. shows the opposite effect [
13
]. As in Fig.
11.9
,
there are three current blockage levels for poly (A) and all of them are actually
smaller than those of poly (C).
a
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