Biomedical Engineering Reference
In-Depth Information
the subsequent assembly of the genome, while eliminating logistically challenging
and error-prone amplification and library formation due to the exquisite single
molecule sensitivity. Moreover, electrical detection of DNA using a nanopore has
several other advantages over luminescent or fluorescent detection - it has more
dynamic range and requires less preprocessing.
12.1.2 The Prospects for Single Molecule Sequencing
with a Nanopore
The nanopore sequencing concept uses a radically new approach to detection that
relies on the electrical signal that develops when a DNA molecule immersed in
electrolyte translocates through a pore in a membrane. By applying an electric field
to a nanopore in a thin membrane, it is possible to force the highly charged
polyanionic DNA molecule to move through it with nucleotides in single-file
sequential order. The electric potential of the nucleotides in a pore presents an
energy barrier to the passage of ions and blocks the current in a characteristic way.
If each nucleotide has a characteristic electrical signature, then a pore could be used
to analyze the sequence, reporting all the signatures in a single long read without
resorting to multiple DNA copies.
The prospect for sequencing DNA with a nanopore is being carefully scrutinized
right now [
11
-
20
]. To sequence DNA using a nanopore requires a robust, porous
structure of an appropriate size - comparable to or even less than the size of DNA -
to maximize the electrical signal. The prototypes being explored fall into two
categories generally: (1) proteinaceous pores such as
-HL) shown
in cross-section in Fig.
12.1a
and its mutants; or (2) nanopores in solid-state
membranes like that illustrated in Fig.
12.1b
. Since the translocation velocity
through the pore can be very high - about 1 bp/10
a
-hemolysin (
a
m
sfor
a
-HL [
12
] and ~1 bp/10 ns
Fig. 12.1 Nanopores for sequencing DNA. (a) A cross-section through an
-HL pore taken from a
simulation showing a trapped DNA hairpin. The pore diameter (1.3 nm) is smaller than the stem of
the hairpin. (b) A cross-section through a solid-state pore in a silicon nitride membrane showing a
double stranded DNA molecule trapped in a 2 nm diameter pore. The diameter of the solid-state
a
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