By Monica Heger
Overcoming a major hurdle for nanopore sequencing, researchers from Arizona State University have designed a sensor that can distinguish all four individual bases of DNA.
Using a technique called tunneling, the bases pass through a tunnel gap with two electrodes. As they pass through, they form unique hydrogen bonds with chemical reagents, producing a current. The team was able to design the tunnel gap so that each base produced a unique current spike.
The study, which was published last week in Nano Letters, builds on previous work by the same team, which published a method for DNA translocation through a carbon nanotube in January (see In Sequence 1/12/2010).
Stuart Lindsay, director of Arizona State's Center for Single Molecule Biophysics at the Biodesign Institute and senior author of the study, told In Sequence that the ultimate goal is to combine this readout with the carbon nanotube nanopore, for a complete device.
The key to the work was setting the tunnel gap just right, and determining which reagents formed optimal hydrogen bonds with the bases. Lindsay said he used computer simulations to determine the four optimal hydrogen bond formations for each base.
"At one particular gap, we could get unique signals," he said. If the gap is too large, the frequency of reads drops, and if it is too small, then you get multiple types of reads, he said. "There's sort of a sweet point in the tunnel gap that lets you read all four bases."
Lindsay tested the device on single nucleosides and achieved accurate reads about 60 percent of the time. Next, he tested mixtures of two nucleosides at once, and found that the reader could distinguish between the nucleosides, which he said was encouraging. When he mixed adenine and guanine, the reader showed two different spikes. When he halved the concentration of adenine, the corresponding current spike decreased. This was an important test, he said, because it showed that the reader was in fact reading single molecules.
"There have been a number of impressive developments in nanopore technology in the last two years — this contribution from the group at Arizona State is a terrific example," Mark Akeson, a professor of biomolecular engineering who also works on nanopore sequencing, at the University of California, Santa Cruz told In Sequence in an e-mail.
Other groups have worked on distinguishing single nucleotides as they pass through a nanopore using other techniques, such as measuring the change in ion blockage as DNA passes through a nanopore, but no group has built a complete functional device (see In Sequence 2/24/2009 and 3/21/2009).
Akeson noted that the accuracy rate would need to be improved before the ASU base-reading method is commercially practical. He added that combining the reader with the carbon nanotube translocation will be a high hurdle. "There's nothing easy about engineering at this scale," he said.
Before Lindsay attempts to build the actual device, he first wants to test the reader on oligonucleotides and then single-stranded DNA to ensure that DNA's structure doesn't affect the reader, and also to verify that each base that passes through is being read, and not just a fraction of them.
The last step will be to combine the reader with translocation through the carbon nanotube. Lindsay said that he hopes to have a prototype in one year. Ultimately, he thinks that the device can be built onto a computer chip.
"This experiment has lots of things that need to be pursued, but the really big news is that yes, you can measure the base composition by tunneling," said Lindsay.