Single molecule sequencing with nanopores

Lots of people have been trying for a long time to use nanopores to sequence DNA molecules (David Deamer at UCSC and Amit Meller at BU being two of the biggest proponents). The basic idea is to electrophoretically force a piece of DNA through a nanopore, and watch how the current changes in order to figure out which bases are moving through the pore. But, there have always been problems in getting this to work at single base resolution, mostly due to the DNA moving too fast through the pore, and other factors which cause the noise to overwhelm the tiny differences in signal when the different bases are in the pore. This has made it unusable for sequencing.

Now, a paper in JACS (along with a review by Vincent Croquette) demonstrates the first sequencing of a DNA using a nanopore. The technique relies on two improvements: first, a short ssDNA molecule is blocked on both ends, by a biotin/streptavidin blockade on one, and by double stranded region on the other, which cannot fit through the pore. This allows the molecule to be cycled back and forth several times through the pore. Second, by attaching a long PEG chain to the single stranded end, a large dielectric gradient is formed at the very end of the single stranded region. When this region is in the pore, the current becomes very sensitive to the position of the PEG/DNA junction in the pore. So, by adding polymerase and DNA bases one at a time, changes in the position of the PEG/DNA junction can be detected when one or more NTPs are incorporated. By flowing in NTPs sequentially, it can be determined what the next base or bases is by which base is present during the next elongation step. This is similar in spirit to the
Quake approach, but the latter used incorporation of fluorescent bases, and TIR imaging. (This approach became the basis of Helicos HeliScope "single molecule sequencer." I don't really know much about how this thing performs in the field, and how many of these they've sold.) The Croquette review also draws comparisons with Will Greenleaf's 2006 paper from our lab demonstrating single molecule sequencing using high resolution optical trapping of RNA polymerase.

This is an interesting technology, and a huge step forward for nanopore sequencing efforts. But, like the optical trapping approach, it has scalability problems, and isn't really ready for prime time.