Nanopore Sequencing Takes More Small Steps

 By Kevin Davies 

December 3, 2010 | New research shows that the decade-long goal of sequencing DNA by passing an intact single strand of DNA through a protein nanopore is edging closer to reality.  

In a paper published in the Journal of the American Chemical Society this month, Mark Akeson, David Deamer and colleagues at University of California Santa Cruz have demonstrated the continuous and controlled translocation of a single-stranded DNA molecule through a protein nanopore using a single DNA polymerase enzyme. This, the authors say, provides the foundation for a molecular motor and an essential component of so-called strand sequencing using nanopores.   

The idea of single strand sequencing, using an enzyme to regulate movement, has been around since early the 1990s, originally developed by Harvard’s Dan Branton and Akeson’s colleague David Deamer.  

“It’s taken a while to demonstrate that this could work,” Akeson told Bio-IT World. “The breakthrough with the phi29 DNA polymerase is that it binds with high affinity, so when you apply force with the electric field, it’s held on very tightly. The breakthrough was we could observe catalysis and the addition of nucleotides to the elongating DNA strand over numerous molecules.”  

Many groups are pursuing various forms of nanopore sequencing using natural and artificial nanopores. British biotech company Oxford Nanopore is arguably the farthest along, having demonstrated in 2009 the ability to distinguish individual bases of DNA – such as could be cleaved with an exonuclease enzyme -- when passed through a bacterial alpha-hemolysin nanopore. While the sales and marketing rights for the exonuclease method have been licensed to Illumina, Oxford Nanopore continues to explore other methods of nanopore sequencing, and has been collaborating with Akeson’s group for a few years to that end.  

“Oxford Nanopore is developing two methods of DNA sequencing,” said Oxford Nanopore CEO Gordon Sanghera. “Both fit to our proprietary array chip-and-reader technology platform. The exonuclease sequencing method is perhaps best known, but in recent months our collaborators have made exciting developments in vital techniques for strand sequencing. Internally, we are working hard with our collaborators on a strand sequencing programme to realise these breakthroughs.”  

Do the Strand  

In strand sequencing, disruption in the electric current across the nanopore are measured and used to discriminate and identify the constituent bases in the DNA molecule as it passes through the pore.   

The new study from Akeson, chair of biomolecular engineering at UCSC, and colleagues, advances work published a few months ago in Nature Nanotechnology that showed that DNA could be moved through a nanopore using a series of polymerases (necessitating some complex electronics). New techniques developed by the UCSC group allow for continuous single-stand DNA movement, providing an uninterrupted signal as the strand travels through the nanopore in real time.   

The new study shows that “DNA translocation control is achievable in conditions that are compatible with an electronic sequencing technology.” Akeson’s group established a rate of DNA translocation (base incorporation) of about one base every 20 milliseconds, or about 50 bases per second. “At the end of the day, that’s both readable and fast enough to be useful,” says Akeson.   

The viral phi29 polymerase allows the DNA strand to be moved very systematically, says Akeson. Besides measurements using ionic current impedance, Akeson says, “One could also imagine also coupling the enzyme to solid state pores and measuring the nucleotide in the pore by different methods.”  

Akeson also stresses the other inherent virtue of this nanopore approach is the theoretical ability to read long pieces of DNA. “The entire field is converging on a device that is likely to read bases and sequence before too long. Whether it’s an important biotech device is going to depend on a lot of other factors.”  

Further Reading: Processive replication of single DNA molecules in a nanopore catalyzed by phi29 DNA polymerase. Available online at http://pubs.acs.org/doi/abs/10.1021/ja1087612 (subscription required)