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Monday, September 27, 2010
By Tim Stephens with contribution by Richard Hughey
Research by Mark Akeson, professor and chair of biomolecular engineering, and his colleagues in the Baskin School of Engineering at UC Santa Cruz shows a new method of enzyme-controlled movement of a single strand of DNA through a protein nanopore. The findings, published in Nature Nanotechnology, represent a key step toward nanopore sequencing of DNA strands.
The publication describes the observation of single-stranded DNA (ssDNA) as it translocates through a protein nanopore, alpha hemolysin (AHL). Movement of the ssDNA was controlled by polymerase-facilitated replication of individual DNA molecules. This movement could be initiated under electronic control.
Polymerase activity was shown to be blocked in solution when the ssDNA was not at the nanopore opening. Capture of the strand by the pore removes a blocking strand of nucleotides and allows the polymerase to function on the trapped strand.
The UCSC researchers are collaborating with Oxford Nanopore Technologies in the development of a new generation of electronic, single-molecule DNA sequencing technology. In the 'strand sequencing' method, current through a nanopore is measured as a DNA polymer passes through that pore. Changes in this current are used to identify the DNA bases on the DNA molecule, in sequence. This paper addresses a key challenge for DNA strand sequencing: fine control of the translocation of the DNA strand through the nanopore, at a rate that is consistent and slow enough to enable accurate identification of individual DNA bases. The Nature Nanotechnology work shows for the first time that the motion of a strand can be controlled using electronic feedback and that an enzyme can move a strand against a field while located on top of the nanopore.
"The techniques described in this paper are an advance towards electronic, single-molecule DNA sequencing," Akeson said. "Electronic control of DNA translocation through a protein nanopore is a scientific goal that we have strived towards for years, and these methods are now forming the basis for further work in our laboratories. We are excited by our collaboration with Oxford Nanopore, whose parallel nanopore sensing strategy is impressive."
Professor Akeson particularly credited the contributions of undergraduates Gerald Max Cherf and Joseph Dahl. Cherf, who received a Chancellor's Award for his undergraduate research, is one of seven members of the first graduating class of the Baskin School's new B.S. in Bioengineering.
Reference: Replication of individual DNA molecules under electronic control using a protein nanopore. Felix Olasagasti, Kate R. Lieberman, Seico Benner, Gerald M. Cherf, Joseph M. Dahl, David W. Deamer and Mark Akeson Nature Nanotechnology September 2010.
DOI: 10.1038/NNANO.2010.177, (subscription needed)