This article was originally published Aug. 26.
By Julia Karow
In a step towards nanopore-based sequencing-by-hybridization, researchers at Brown University have shown that they can use a solid-state nanopore to distinguish double-stranded from single-stranded regions in a DNA molecule translocating through the pore.
According to the scientists, who described their findings in Nanotechnology last month, this is the first published example of using a solid-state nanopore to resolve short double-stranded probes on single-stranded DNA by measuring the ionic current.
Senior author Sean Ling, a professor of physics at Brown, told In Sequence that the aim is to use nanopores to detect the location of known hybridization probes on DNA, and to reconstruct the DNA sequence from the results. This, he said, is easier than resolving individual bases of DNA translocating through the pore, an approach pursued by many other groups working on DNA nanopore sequencing.
Ling is a co-founder of Nabsys, a startup that is developing the hybridization-assisted nanopore sequencing, or HANS, approach commercially, and a co-inventor of the firm's technology. He said he had a falling out with the company's management, however, and is no longer on its board, noting that his group and Nabsys are now "friendly competitors."
For their study, the Brown scientists constructed three oligonucleotides, each about 140 bases in length, where the first one hybridizes at one end with the second, and the second hybridizes at its other end with the third. The result is a single-stranded DNA trimer, about 400 bases in length, with two 12-base double-stranded segments that are separated by about 140 bases.
In order to slow down the movement of the DNA, they attached a polystyrene bead to one end of the DNA. Then they measured the ionic current as the other end of the DNA passed through a solid-state nanopore, which they had fabricated by drilling through a silicon nitride membrane with a transmission electron microscope.
The researchers observed a characteristic "double dip" in the ion current, which they attributed to the presence of the two 12-mer double-stranded regions. These results showed "remarkable agreement" with a computer simulation, Ling said. "What we claim is that we succeeded in using a nanopore to detect a hybridization probe."
However, 12-mer hybridization probes would be unsuitable for hybridization-based sequencing, he explained, because that would require 16 million different probes. The reason his team used 12-mer regions is that unlike shorter ones, they are stable at room temperature. For shorter hybridization probes, they would need to use a cooling stage. Ling's next goal is to cut the length of the double-stranded regions in half, to 6-mers, and to detect those also by nanopores. "If we can do that, then we should be in business," he said.
Eventually, he wants to combine the nanopore-based probe detection with a magnetic tweezer approach to slow down the DNA, which his group described in a paper a year ago (IS 4/21/2009).
Nabsys executives declined to comment on the Brown group's study. The company has not yet published its own results in a journal. But at a meeting organized by the National Human Genome Research Institute earlier this year, company researchers showed that they can use solid-state nanopores to detect up to three oligo probes hybridized to single-stranded DNA coated with the RecA protein in order to slow the molecule down and to improve the signal-to-noise ratio. In addition, they said they have been able for a while to detect probes hybridized to double-stranded DNA, forming regions of triplex DNA (IS 3/16/2010).