By Monica Heger
Building on previous work towards single-molecule sequencing using a solid-state nanopore sensor and optical readout, researchers from Boston University took a step towards making the DNA capture portion of the technique more efficient. Last month, they published their results in Nature Nanotechnology.
Boston University physicist Amit Meller, who first suggested the technique (see In Sequence 08/08/2006), made the DNA capture more efficient by applying a salt gradient across the nanopore. The salt gradient produced an electrostatic effect, drawing the DNA to the nanopore. Meller and colleagues said in the paper that the application of the salt gradient enabled them to use about 10,000 times less starting DNA than they could previously, and also improved the capture rate for longer strands of DNA and increased average translocation times compared to prior implementations of the method and other methods using solid-state nanopores.
"What we're showing is that the nanopores can be made much more sensitive by applying a special electrostatic focusing effect," Meller told In Sequence. The next step will be to pair the capture method with the sequence-reading method.
Meller said that the increased sensitivity allowed his team to use much less starting DNA, which reduced the need for numerous amplification steps. Eventually, Meller said, no amplification will be needed. "We reduced the copy number to a point where we think that in the near future, we will not need any PCR. You [will be able to] start with genomic material directly from the cells," he said. Not having a PCR step will reduce the cost, speed up the process, and make it more accurate, he added.
"It's a very important technological advance," said David Deamer, a chemist at the University of California, Santa Cruz, who works with biological nanopores. "When we work with nanopore analytical methods in our lab, we need to use micromolar concentrations of DNA to get enough signals to analyze. What [Meller] has shown is that it's possible to use salt gradients and a synthetic nanopore to capture picomolar amounts of DNA — a million fold improvement in sensitivity [over our nanopores]," he added.
Aside from reducing the amount of needed sample, the salt gradient also increased the time it took for the DNA to pass through the pore, which will make it easier for the sensor to read the sequence as the DNA goes through the pore. Meller said it wasn't quite clear why the translocation was slower, but he thinks that the interaction between the positive ions from the salt gradient and the negatively charged DNA cause something akin to a drag force, slowing down the DNA.
Another somewhat surprising result, according to Meller, was that the longer DNA molecules were more efficient at threading through the nanopore than shorter molecules. "Intuitively you'd expect a longer length spaghetti would have a lower chance of finding and entering the pore" because it is heavier and the end would have a lower probability of finding the pore, Meller said. Nevertheless, they found the opposite was true.
Because there is an electric field channeling the DNA molecules towards the pore, and because the larger DNA molecules have a greater charge, they will feel the effects of that electric field from farther away than shorter molecules, Meller said. Then, once they reach the pore, the stronger field holds them at the pore, increasing the likelihood that an end of the DNA fragment will enter the pore.
Meller said he and his colleagues found that the capture rate grows with molecular weight up to about 10,000 base pairs, and then plateaus. However, he thinks that with a few improvements, the effect could be seen in DNA fragments up to 50,000 base pairs. "And, if we can sequence these kinds of long DNA, that will be a really big breakthrough," he said.
While the capture method could potentially be compatible with different sequence-reading methods, it may be limited to synthetic nanopores, said Deamer. Synthetic nanopores are much more stable than biological nanopores. Biological nanopores are built into a lipid bilayer, which may not be able to withstand the conditions produced by the salt gradient. "The salt gradient might pop the membrane," Deamer said. One potential way to get around that problem, he added, would be to use a hybrid approach — a biological pore inserted into a synthetic membrane.
Meller, who is a member of Oxford Nanopore Technologies' technical advisory board, said he is now working on commercializing the technology. His previous work on solid-state nanopore sequencing was licensed by Sequenom (see In Sequence 10/02/2007). He said that original agreement is still in place, but would not comment on other potential partners or future licensing deals.