The second generation of DNA sequencing machines have now taken over the market, and their high throughput is producing new genomes at a staggering clip. But the third generation of machines are already in the development pipeline, with features like longer DNA reads, faster speeds, and single-molecule precision. Over the weekend, Nature Nanotechnology published a paper on one of the promising technologies that's currently in the works: nanopore sequencing. It reports some preliminary success in developing a computerized nanopore system that controls when DNA bases are added, and reads them one-by-one in the process.

The term "nanopore" would seem to imply a bit of carefully structured metal; it's anything but. The pore in question is simply a protein that embeds in a membrane and creates a tiny passage through it, just big enough to fit a single strand of DNA. Since DNA carries a negative charge, applying a voltage across the membrane can drive a single strand of DNA through the pore (the double helix won't fit); as it travels through, small voltage changes result that can be used to "read" the sequence. The problem is that the molecules tend to fly through too fast for a clear signal to be picked up, so various tricks are being considered in order to slow things down, like chewing up a DNA molecule one base at a time.

The new paper suggests that it might be possible to use the same voltage differences used to send the DNA through the pore to control how quickly bases are added to DNA by an enzyme called polymerase, which normally copies the molecule (we've described DNA polymerases in detail previously). The process starts by getting a polymerase to stick to a strand of DNA, ready to make a copy of it. When a voltage is applied, the DNA moves through the pore, but the polymerase is too big, and gets stuck on the outer surface.

When it's jammed up against the surface of the pore, the polymerase can't add any bases; the whole system is stuck. That's where electronics come in. The authors set up a field programmable gate array to run a Finite State Machine that steps the system through a cycle. The first step is to lower the voltage difference, which lets the DNA snake backwards through the pore. This frees the polymerase, which takes a few milliseconds to add a base. The voltage is then reapplied, and the system senses whether a new base has been added, creating a longer stretch of double helix and "pulling" the single stranded section back out of the pore, one base at a time. With a new base stuck in the pore, it should be possible to read it.

Right now, the system has a lot of rough edges: it misses some bases, only works for short stretches of DNA, and doesn't discriminate between bases very well. But the authors claim that there are ways that all of these issues could be improved. And, even if it doesn't beat some of the alternatives to market, it's a very interesting mix of biochemistry and electronics.

Nature Nanotechnology, 2010. DOI: 10.1038/NNANO.2010.177 (About DOIs).