Third Generation DNA Sequencing with a Nanopore - Nanopores: Sensing and Fundamental Biological Interactions - page 293

12.1.3 Chapter Overview

Nanopores promise a new paradigm not only for DNA sequencing, but for
polynucleotide synthesis, and even information processing and computing. How-
ever, that promise won't be realized unless the translocation kinetics and the
molecular configuration in the pore are stringently controlled. In this chapter,
following several recent reviews, [ 33 - 35 ] we will narrowly focus on recent
developments [ 21 ] concerning the prospects for sequencing double-stranded
DNA using a synthetic nanopore with a diameter less than the double helix.
Our plan is to use this example to illuminate both the challenges and some of the
available solutions. First, we illustrate how state-of-the-art in semiconductor
nanofabrication can be applied to produce a sub-nanometer diameter nanopores
in heterostructure membranes, and tailor the geometry to the propeller-like
structure of DNA. Thus, semiconductor nanotechnology plays a key role in
our plan for sequencing a single DNA molecule.
A nanopore is sensitive to single molecules DNA. But to take full advantage of
the minimal material requirements, we have to somehow convey the DNA to the
pore. We show two schemes that use microfluidics as a molecular conveyance for
DNA: hydrodynamic focusing and integrated valves in nanofluidic circuits. Work-
ing separately or together, these strategies could be used to force the DNA to within
the capture radius of the electric field extending outside the nanopore. Once it is
captured by the electric field, the translocation kinetics through the pore has to be
controlled to ensure signal fidelity. We have developed a new method for trapping a
single molecule of dsDNA in a nanopore, smaller in diameter than the double helix.
We show that it is possible to trap a single dsDNA molecule in a nanopore

3nmin
diameter by first applying a voltage larger than the stretching threshold, forcing the
molecule to translocate through the pore. According to molecular dynamics (MD)
simulations, this leaves the dsDNA stretched in the pore constriction with the base-
pairs tilted, while the B-form canonical structure is preserved outside the pore. If
the electric field is rapidly switched to a value below the threshold during the
translocation, a single dsDNA molecule becomes trapped in this configuration, in
what is effectively a harmonic potential. In principle, the molecule can subse-
quently pushed through the pore one base at a time, provided the voltage forcing
the DNA through the pore is switched fast enough. If the duration in the trap is
commensurate with the bandwidth, we further assert that we can discriminate
distinct signatures of C-G and A-T base-pairs, under less than optimal conditions,
by simply measuring the pore current.
We envision using this type of trap in a sequencing protocol whereby the
translocation kinetics of dsDNA in a pore are stringently controlled and mea-
surements are performed to extract the identities of the nucleotides from the pore
current. One obvious problem with sequencing this way is determining which
nucleotide is on which strand, e.g. distinguishing A-T from T-A. And so, finally
we consider the limitations imposed on the SNR by synthetic nanopores currently in
production. We show that it should be possible to call base-pairs with sufficient

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