Oxford Nanopore moves closer to label-free, single molecule DNA sequencing

23 February 2009

Oxford Nanopore Technologies has announced the publication of new research in Nature Nanotechnology, demonstrating accurate and continuous identification of DNA bases using nanopores. The system can also directly identify methylated cytosine, which is important in the study of epigenetics.

This research marks significant progress towards Oxford Nanopore’s goal of developing the first label-free, single molecule DNA sequencing technology.

A method of identifying single molecules that does not rely on expensive and complex fluorescent labelling is central to achieving the next dramatic improvement in the cost and speed of genome analysis.

It is possible to achieve this goal by monitoring a simple electrical current passing through a nanopore. As single DNA bases pass through the nanopore, each base causes a characteristic disruption of current that allows the molecule to be identified.

Today’s Nature Nanotechnology paper describes the use of nanopore technology to identify DNA bases with very high confidence for integration into a highly competitive new DNA sequencing system. Results were achieved through modification of a protein nanopore, including the permanent attachment of an adapter molecule to its inner surface.

The publication also demonstrates that methylated cytosine can be distinguished from the four DNA bases using the same method. This is important in the study of cancer, where genome methylation is implicated but existing study techniques require complex labelling of the DNA sample.

“The science of nanopore DNA sensing is now accurate and reliable enough to support a breakthrough industrial technology,” said Professor Hagan Bayley, founder of Oxford Nanopore and an author of the paper.

“The simplicity and versatility of nanopores as a sensing system has intrigued academic researchers for nearly two decades. We anticipate that with the fast pace of the science, nanopore devices will soon be used for the measurement of DNA and many other molecules.”

“The findings from this paper provide validation of the high performance of the nanopore-sensing element of our DNA sequencing system,” said Dr Gordon Sanghera, CEO of Oxford Nanopore Technologies. “We continue the rapid development of this technology, whose advantageous cost, speed and versatility promise to enable a new paradigm of DNA analysis.”

The research includes other outcomes essential for an integrated nanopore sequencing system. For its BASE Technology, Oxford Nanopore couples a protein nanopore with a processive exonuclease enzyme. This enzyme cleaves individual DNA bases from a strand of DNA and introduces the individual bases into the nanopore for identification.

This study shows that the operating conditions of the nanopore are compatible with those of an exonuclease. In addition there is a high probability that each DNA base translocates the nanopore so that a base is not read twice.

Discrimination of nucleotides:

The accuracy of identification of DNA bases was determined as follows: The distributions of bases as dNMPs were fitted to Gaussians and the areas of peak overlap were determined to give confidence values for base identity.

The percentages of binding events that could be assigned to each base with a confidence approaching 100% at a high salt concentration (800 mM) were 99.9% (G), 99.7% (T), 99.8% (A) and 99.99% (C). Where there is ambiguity in a base call, the identities of the only two possible alternative bases are known.

This research was concluded in the summer of 2008 at the University of Oxford and Oxford Nanopore, where scientists continue to improve on this performance.

1. James Clarke, Hai-Chen Wu, Lakmal Jayasinghe, Alpesh Patel, Stuart Reid1 & Hagan Bayley. Continuous base identification for single-molecule nanopore DNA sequencing. Nature Nanotechnology. Published online: 22 February 2009 | doi:10.1038/nnano.2009.12
The abstract is available at: http://www.nature.com/nnano/journal/vaop/ncurrent/

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