Nanopore sequencing
Nanopore sequencing has the potential to offer relatively low-cost genotyping, high mobility for testing, and rapid processing of samples, including the ability to display real-time results.
In 1989 he sketched out a plan to push single-strands of DNA through a protein nanopore embedded into a thin membrane as part his work to synthesize RNA.
In 1999 they published the first paper using the term 'nanopore sequencing' and two years later produced an image capturing a DNA hairpin passing through a nanopore in real time.
[15] Applying a bias voltage across the membrane induces an electric field that drives charged particles, in this case the ions, into motion.
Inside the capture region, ions have a directed motion that can be recorded as a steady ionic current by placing electrodes near the membrane.
A nano-sized polymer such as DNA or protein placed in one of the chambers has a net charge that feels a force from the electric field in the capture region.
[14] Inside the pore the molecule occupies a volume that partially restricts the ion flow, observed as an ionic current drop.
Based on various factors such as geometry, size and chemical composition, the change in magnitude of the ionic current and the duration of the translocation vary.
Sequencing was made possible because passing through the channel of the nanopore, the samples cause characteristic changes in the density of the electric current.
[18] Sufficiently low translocation velocity can be attained through the incorporation of various proteins that facilitate the movement of DNA or RNA through the pores of the lipid membranes.
[19] Alpha hemolysin (αHL), a nanopore from bacteria that causes lysis of red blood cells, has been studied for over 15 years.
A recent study has pointed to the ability of αHL to detect nucleotides at two separate sites in the lower half of the pore.
[25] The R1 and R2 sites enable each base to be monitored twice as it moves through the pore, creating 16 different measurable ionic current values instead of 4.
The natural nanopore was modified to improve translocation by replacing three negatively charged aspartic acids with neutral asparagines.
[28] The electric current detection of nucleotides across the membrane has been shown to be tenfold more specific than αHL for identifying bases.
These studies were conducted using a scanning probe microscope as the sensing electrode, and have proved that bases can be identified by specific tunneling currents.
[32] After the proof of principle research, a functional system must be created to couple the solid state pore and sensing devices.
An effective technique to determine a DNA sequence has been developed using solid state nanopores and fluorescence.
When the dsDNA is translocating through a solid state nanopore, the probe strand will be stripped off, and the upstream fluorophore will fluoresce.
One example is that a motor protein may only unzip samples with sufficient speed at a certain pH range while not operating fast enough outside of the range- this constraint impacts the functionality of the whole sequencing unit.
Both of these examples would have to be controlled for in the design of any viable biological nanopore system- something that may be difficult to achieve while keeping the costs of such a technology as low and as competitive, to other systems, as possible.
In the early papers methods, a nucleotide needed to be repeated in a sequence about 100 times successively in order to produce a measurable characteristic change.
As of 2006, the problem has been tackled by either improving the recording technology or by controlling the speed of DNA strand by various protein engineering strategies and Oxford Nanopore employs a 'kmer approach', analyzing more than one base at any one time so that stretches of DNA are subject to repeat interrogation as the strand moves through the nanopore one base at a time.
