By Julia Karow
This article was originally published March 24.
Two nanopore research groups — led by Jens Gundlach at the University of Washington in Seattle and Mark Akeson at the University of California, Santa Cruz — have joined forces to enable DNA strand sequencing.
Under a collaboration, the teams are combining the MspA protein nanopore, which the UW group has been exploring for base recognition, with phi29 polymerase, which the UCSC group has harnessed to control the speed of DNA moving through a pore.
"It's a full sequencing system — all we have to prove is that we can operate phi29 together with MspA," Gundlach told In Sequence. "If we achieve that, that will be a very significant result."
Both groups stress that their collaboration is funded by government grants and is independent of Oxford Nanopore Technologies, which holds an exclusive license to certain nanopore technology developed in Akeson's laboratory at UCSC — some in partnership with Harvard University — and funds a portion of his research. Akeson has also been a member of Oxford's technical advisory board since 2008.
Gundlach said the University of Washington has not licensed the MspA pore technology to any company yet, although several have expressed an interest. Oxford Nanopore has focused on another protein nanopore, alpha-hemolysin, which has been extensively studied by its founder and collaborator, Hagan Bayley at the University of Oxford.
The UW and UCSC groups are tight-lipped about the status of their experiments and any preliminary results, fearing they might get scooped in a field that has become fiercely competitive over the last few years as more researchers have turned their interest to nanopore sequencing (IS 1/11/2010).
However, "we are relatively confident that this will work," Gundlach said. His guarded optimism is based on the fact that two limitations to nanopore sequencing have recently been overcome.
One is the ability of a nanopore to read the DNA sequence with single-nucleotide resolution. Last summer, Gundlach's team published a proof-of-principle study in which they showed that an engineered MspA can distinguish all four nucleotides in single-stranded DNA and resolve single nucleotides when stretches of double-stranded DNA are inserted between them (IS 8/24/2010).
Likewise, Bayley's group showed last summer that they can recognize the four different bases in single-stranded DNA using alpha-hemolysin after "sharpening" one of the pore's three base-recognition sites (IS 10/12/2010).
The other limitation has been the high speed at which DNA translocates through a nanopore when a voltage is applied — too fast to record signals for every nucleotide. One way to work around this is Gundlach's approach to insert double-stranded DNA between each base, or "duplex-interrupted" sequencing, which his group is still pursuing and has recently advanced with "some really interesting results," he said.
Another way is to use DNA-processing enzymes, such as a polymerase, to slow down the DNA. Akeson's group showed at the end of last year that phi29 polymerase can replicate DNA that is trapped inside a nanopore and slow down its translocation, an improvement of earlier work (IS 9/28/2010).
"As soon as we saw that they're making great progress on this enzyme-actuated nanopore sequencing, we figured it would be foolish for us to compete with them, but rather join forces," said Gundlach, who maintains that MspA is better suited than alpha-hemolysin for nanopore sequencing because it delivers stronger and more distinct current signals. "It's just a great combination."
Irrespective of who will provide the winning combination for DNA sequencing by nanopores, both Gundlach and Akeson believe it will happen soon. "I would be surprised if one or more academic labs did not publish short DNA strand reads using nanopores in the coming year," Akeson told In Sequence.
But while short-read sequencing with nanopores is "almost a done deal," he said during a talk at the Advances in Genome Biology and Technology conference last month, "the big issue is getting long reads with this device."
He and his colleagues are focusing on "important technical questions" such as read length and enzyme processivity as a function of voltage, he said.
"I think there is a realistic chance that nanopores will, in the next year or so, really demonstrate proof-of-concept," Gundlach said. "And in the next couple of years, a system could come on the market that really uses nanopores."
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