The ability to sequence the human genome rapidly (e.g. in 15 minutes) and at an affordable price (e.g. US $1000) would be a turning point in medicine. At that price and speed, most people could afford to have their genome sequenced on-the-fly for tailored medical treatment. Thanks to advances in second-generation DNA sequencing techniques, the cost of sequencing an entire human genome is now about US$ 50,000.1, 2
However, most second-generation technologies such as those implemented by 454 Life Sciences (Roche), Solexa (Illumina) and Applied Biosystems SOLiD™
(Life Technologies), rely on slow iterative cycles of enzymatic processing and imaging-based data collection. Therefore, the most likely candidates to cross the US $1000 and 15-minute human genome barrier will be third-generation single-molecule sequencing platforms, such as Pacific Bioscience’s single molecule-real time sequencing by synthesis (SMRT™
) or nanopore sequencing,3
which are based on the continuous determination of sequences of individual DNA molecules by cycle-free processes.
In one approach to nanopore sequencing, a single-stranded DNA or RNA molecule is driven electrophoretically through a nanopore and each DNA base is read as it passes a recognition point.3
In its simplest manifestation, the current associated with the passage of ions (e.g. K+
) through the nanopore during DNA translocation provides the electrical read-out required to distinguish each base. In our laboratory, we use α-hemolysin (αHL) protein nanopores reconstituted in planar lipid bilayers (). Notably, we have shown that the four DNA bases,4–6
including their epigenetically modified forms, 5-methylcytosine and 5-hydroxymethylcytosine,7
can be discriminated in a DNA strand immobilized within the nanopore.
Figure 1 Section through a 7R7 nanopore. The amino acids at positions 113, 115, 117, 119, 121, 123 and 125 were replaced in the WT7 nanopore (PDB:7AHL) by arginine by using PyMOL software (DeLano Scientific LLC, v1.0). 7R7 is in the RL2 background, in which lysine (more ...)
Alternatively, single nucleotides are identified by reading the tunnelling current passing through individual DNA bases, while a DNA strand is translocating through a solid-state nanopore modified with tunnelling probes.8–10
Tunnelling readings would be advantageous because the nano-ampere range of tunnelling currents will allow the reading of nucleotides at a greater speed than with the pico-ampere ionic currents through protein nanopores.3
In addition, since the tip of the probe can be less than 1 nm in diameter,3
a single nucleotide would be addressed by the tunnelling probe at any given time.
One of the main remaining challenges in nanopore sequencing is to reduce the speed of DNA translocation through the nanopore, which is too fast to allow discrimination between individual nucleobases by using either ionic11
currents. Previous attempts to reduce the speed of free DNA translocation have included the use of low temperatures12
and increased viscosity13, 14
. These approaches reduced the speed of DNA translocation by one order of magnitude or less, at the expense of a large decrease in the ionic current. In this work, we show that by lining the transmembrane region of the αHL pore with positively charged residues, the speed of DNA translocation can be slowed by more than two orders of magnitude. Although, the ionic current during DNA translocation is again almost completely suppressed, we suggest that such nanopores could be used to control the speed of DNA translocation in hybrid protein and solid-state nanopores.