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At NHGRI Meeting, Nanopore Groups Report First Successes in Reading DNA

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By Julia Karow

Several research groups last week reported progress towards nanopore-based DNA sequencing, with one group being able to read several bases electronically from a single modified DNA molecule, and another team reading out bases from converted DNA using an optical method for the first time.

At the National Human Genome Research Institute's Advanced DNA Sequencing Technology Development meeting in Chapel Hill, NC, researchers from the University of Washington, Nabsys, and Boston University reported significant advances in electronic and optical approaches to nanopore sequencing.

All three groups have received funding under the NHGRI's "$1,000 Genome" grant program.

Jens Gundlach from the University of Washington showed that his group has been able to read a short sequence of bases directly off a single modified DNA molecule passing through an MspA protein nanopore by measuring base-specific variations in the ion current, possibly the first group ever to do so. The researchers had to slow the DNA down in order to make their measurements, which they achieved by inserting double-stranded sections of DNA between the single-stranded bases they wanted to read.

Gundlach explained that he and his group chose to work with MspA — an outer membrane protein from Mycobacterium smegmatis — because of its robustness, its unique shape with a narrow and short constriction that is "ideal for sequencing," the availability of a crystal structure, and because it is cheap to produce and easy to mutate.

In late 2008, they published a paper in which they showed that DNA can pass through a mutated version of the protein pore (see In Sequence 1/13/2009).

Since then, they have demonstrated that each type of DNA base produces a characteristic ion current signal when it passes by a specific site in the protein pore, which can be distinguished even in a random background of bases of DNA that is held in place in the pore. Currents between bases differ by up to tens of picoamps, which Gundlach said "in this business, are enormous differences."

When the DNA moves through the pore, it travels too fast to measure signals. While modifying the DNA is one solution, long-term, he and his colleagues want to read unmodified DNA with single-base resolution. There are other ways to slow the molecule down, such as further engineering of MspA, changes in temperature or viscosity, or mechanical stoppers, Gundlach said. In addition, researchers could use higher-bandwidth electronics, he suggested, adding that his group has already developed a better amplifier.

Gundlach told In Sequence that no company is currently developing MspA sequencing commercially, although he has seen a great deal of interest from several firms.

Optipore DNA Sequencing

While the Seattle group has been pursuing an all-electronic approach, Amit Meller's team at Boston University and his collaborator Zhiping Weng at the University of Massachusetts have been working on a nanopore method with an optical readout, dubbed "optipore DNA sequencing," where fluorescently labeled probes are stripped off the molecule as it passes through a nanopore and their signals recorded by a CCD camera.

Optical detection, Meller explained, is a straightforward way to detect signals from several solid-state nanopores in parallel at high speed, noting that his group showed several years ago how to fabricate an array of nanopores.

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Over the last year, his team has made progress in three areas of optipore sequencing. In order to read DNA, the researchers need to convert it into a larger DNA molecule first, where each nucleotide is represented by two DNA 16-mers, using a binary code. Not only is each base thus "enlarged," but the converted DNA is also less likely to form a secondary structure that would interfere with the readout, Meller said. In a proof-of-concept experiment, the researchers have recently shown that the DNA conversion works, enabling them to convert up to four nucleotides per cycle through a series of steps, including hybridization, ligation, and restriction cleavage, using a library of probes.

Meller's team has also been able to capture long DNA molecules into small nanopores and to increase the capture rate using a salt gradient, a finding they recently published (see In Sequence 1/5/2010).

Finally, the researchers recently demonstrated that they can determine the sequence of DNA from the converted molecule, using a two-color optical readout method. For that, they hybridized probes, so-called molecular beacons, with two types of fluorescent labels to the converted DNA. Each molecular beacon has a quencher, but when the nanopore unzips the probes, they give rise to "a sequence of flashes of light" that provide a signal. The ratio of the color provided from the two kinds of fluorophores determines the identity of the base, Meller said, and the confidence "compares favorably with other single-molecule methods."

His team has also been able to record signals from up to three nanopores simultaneously, he noted.

A number of challenges lie ahead, according to Meller, including automating the process, fabricating high-density nanopore arrays, simplifying the DNA conversion, and switching over to a four-color system.

Nabsys: Single-Base Resolution Not Required

John Oliver from Nabsys reported on his company's progress with hybridization-assisted nanopore sequencing. Instead of reading DNA passing through a solid-state nanopore with single-base resolution, Nabsys is working on a method that detects probes hybridized to the DNA that provide relative positional information and allow the DNA sequence to be reconstructed at the end.

Oliver said that a year ago, the company, which recently raised $7 million in new equity financing, achieved proof of principle. They passed double-stranded DNA through the pore that had regions of triplex DNA where probes had hybridized, and were able to detect the position of the probes electronically.

Since then, he said, the company has been able to detect signals from single-stranded DNA with hybridized probes, using RecA — a single-strand binding protein that coats the DNA — to slow the molecule down and to improve the signal-to-noise ratio "dramatically."

Nabsys has been using up to three probes at once, separated by about 1 or 2 kilobases — for example, probes bound to the 7-kilobase M13 phage genome.

The probe distances calculated from the data are accurate enough to be able to assemble genomes eventually, Oliver said, noting that the DNA accelerates as it travels through the nanopore. The method does not require detectors that record the position of the probes with single-base resolution, he said.

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