Biomedical Engineering Reference
In-Depth Information
[
21
] for a pore in a silicon nitride membrane - it may be possible to sequence a
DNA molecule quickly, driving the cost to practically nothing if the bases can be
discriminated electrically.
But single base resolution on a translocating strand
has not been demonstrated yet.
High fidelity reads demand stringent control over both the molecular
configuration in the pore and the translocation kinetics. Control of the molecular
configuration determines how the ions passing through the pore come into contact
with the nucleotides in the constriction, while the translocation kinetics affect the
time interval in which the same nucleotides are held in the constriction and data is
acquired.
Until now, none of the nanopore prototypes proffered for sequencing has
shown any prospect of satisfying both of these specifications at the same time.
Bayley et al. [
19
,
20
,
22
] recently engineered
-HL in such a way to improve the
SNR, i.e. to hold a nucleotide in place for a longer period of time, in order to
perform more averaging. By modifying the
a
a
-HL such that a cyclodextrin is placed
in the
-barrel, the time that the pore is occluded by a single nucleotide can be
extended. This allows for more accurate determination (
b
90%) of which nucleotide
is in the pore based on blockade current. This method was used in combination with
an exonuclease to determine the composition of single-stranded DNA (
ssDNA
)by
cleaving off individual nucleotides, then measuring them in an
>
-HL pore. This
scheme could potentially be used on raw, genomic DNA. However, it suffers from
the crippling problem of logistics: i.e. how to transport the cleaved nucleotides
from the exonuclease to the pore, ensuring that they arrive in the same sequence as
found in the original DNA, that none escape (missing a base), and that the
exonuclease does not outpace the pore. Tethering the exonuclease to the
a
-HL
has been proffered as a solution, but this scheme is a nontrivial extension to the
original concept.
There are other limitations to using
a
-HL for sequencing. First are the obvious
structural limitations - the protein structure is difficult to change in a predictable
way. Though it is possible to introduce subtle mutations into the protein, gross
structural changes are inordinately difficult. Chief among these structural limita-
tions in
a
a
-HL are the length of the nanopore, and hence the thickness of the
membrane, and the diameter of the pore. For example, the
a
-HL channel limiting
aperture is only 1.5 nm in diameter and several nucleotides long. The secondary
structure of
ssDNA
along with the shape and length of the nanopore obfuscate the
interpretation of a long read, making it practically impossible to measure only one
base at a time. The lipid bilayer presents yet another limitation. The lipid bilayer is
typically 25-100
m in diameter and only 5 nm thick. It ruptures after a few hours
of use or after cycling the electrolyte a few times and the large size of the membrane
produces a capacitance that adversely affects the frequency and noise performance
In contrast
m
-HL, in a solid-state membrane, the pore geometry, the
thickness and composition of the membrane can all be controlled with sub-
nanometer precision using semiconductor nanofabrication practices. This aspect
represents a promising new approach to the creation of a pore. This is important
because the precision translates directly into control of the distribution of the
electric field. The control of the electric field on this scale has already been used
to
a
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