By Will Soutter
Development of Nanopore Technology
Other Next Generation Sequencing Techniques
The ability to determine the sequence of base pairs in a strand of
DNA is one of the most important techniques available to biomedical
researchers. The most common techniques used today were first developed
by Frederick Sanger in the 1970s.
Whilst well established, Sanger techniques remain costly and
complicated to perform, which has limited their application beyond
More recently, various "next generation" sequencing technologies
have emerged, which aim to increase the throughput and accuracy of DNA
sequencing. This will allow sequencing to be applied in more widespread
applications, particularly in the field of personalized medicine.
Some of these novel technologies rely on the understanding that
nanotechnology research has provided. The ability to manipulate
macromolecular DNA structures on the nanoscale has been exploited to
create techniques which can sequence DNA much more rapidly than
conventional Sanger techniques.
Nanopore sequencing is the result of the miniaturization of sequencing
technology down to the molecular level. DNA molecules are threaded
through nanopores, usually either constructed from proteins or etched
into silicon surfaces using nanofabrication techniques.
As the DNA strand passes through the nanopore, the current passing
across the pore depends on which base is passing through at that
moment. Monitoring the changes in current therefore produces a map of
the base sequence.
Under normal circumstances, the DNA strand passes through the pore
too quickly to obtain an accurate reading of the structure. Methods
have therefore been developed to slow down the movement of the DNA
molecules. These methods include attaching various chemical units to
the DNA or to the pore to anchor the molecule, or to increase its
affinity for the pore.
A more advanced technique which has emerged recently is to use DNA
polymerase enzymes to drive DNA through the nanopore at a readable
speed as it is synthesized. This limits the rate of DNA transfer
through the nanopore to the speed at which it is processed by the
enzyme - typically around 10ms per nucleotide, which is slow enough for
the circuitry to gain an accurate reading of the ion current.
Figure 1. This video from Oxford Nanopore
Technologies demonstrates their modular nanopore analysis systems,
which can be calibrated to sequence DNA, RNA, proteins, and other
Development of Nanopore Technology
One of the major promises that nanopore sequencing has made is to
increase the read length - the number of base pairs that can be read
from a single sample of DNA. This is a major limitation of conventional
sequencing technologies, and would allow for much simpler sequencing,
with less sample preparation and higher throughput. There is a
challenge, however, is developing nanopore technology to take full
advantage of its potential. There are many practical issues around high
read-length sequencing which must be overcome, particularly if the
technology has to be scaled to allow readily available, portable
Another challenge which must be met before the next generation
sequencing can be used in all the areas it has potential for is the
nanopore fabrication. This is currently done with a number of different
methods on a research scale, but many of the manufacturing techniques
are not easy to scale up. Even the more conventional techniques like
silicon nanofabrication have to stand up to the rigour of large-scale
production equipment before the full benefits of nanopore sequencing
will be achievable.
in Other Next Generation Sequencing Techniques
Nanotechnology has also informed the development of alternative
sequencing methods, although these have not received the same level of
research attention as nanopore sequencing.
These other nanoscale sequencing techniques include diffusion of
nucleotides labelled with fluorescent chemical tags through a zero-mode
waveguide (an optical trap which is smaller than the wavelength of the
light used in all three dimensions), or using DNA polymerases modified
with quantum dots.
These techniques could drastically improve the throughput and
efficiency of sequencing, although they are effectively improvements on
existing Sanger techniques, rather than new approaches like nanopore
Figure 2. This video from Pacific
Biosciences explains their proprietary technology, using
fluorescent tags to sequence DNA as it is synthesized by DNA polymerase.
Next generation sequencing technologies - in particular nanopore
sequencing - have the potential to revolutionize the field of genomics,
and the wider world of medicine. Unlike many other novel
nanotechnologies, the barriers and challenges which may slow the
commercial adoption of nanopore sequencing appear to be surmountable.
Indeed, some companies, like Oxford Nanopore Technologies and Pacific
Biosciences, have already brought nanopore and other next-generation
- "Briefing: Next Generation Sequencing" - EU ObservatoryNano
- "The potential and challenges of nanopore sequencing" -
Branton et al, Nature Biotechnology 2008. DOI: 10.1038/nbt.1495
- "DNA sequencing with nanopores" - G.F. Schneider and C.
Dekker, Nature Biotechnology 2012. DOI: 10.1038/nbt.2181