Speed-Reading DNA Inches Closer

A British company has demonstrated an important step for a new sequencing technique.

For DNA sequencing to become a routine part of patient care, it needs to become cheaper and faster. A company called Oxford Nanopore hopes to bring down both the cost and the time required for sequencing using a technique called nanopore sequencing. The company has now made an important demonstration of its technology: for the first time, researchers were able to identify DNA bases with near total accuracy. In addition to identifying the four bases of DNA, the technique can also detect a modified version of one of the bases, which may be responsible for causing cancer and other diseases.

Speed reader: A DNA base (red) passes through a protein tunnel lined with a sugar (blue and green bubbles). The sugar slows down the DNA as it moves through the pore, allowing time for the base to be identified.

The new technique allows for the direct identification of bases without the fluorescent labels and imaging equipment used for conventional high-speed sequencing. Direct reading of DNA should not only be faster and cheaper, but it should also make it possible to perform more complex analysis, says Jeffrey Schloss, program director for technology development at the U.S. National Human Genome Research Institute. The Oxford Nanopore system’s ability to detect the DNA modifications catalogued by an emerging field called epigenetics is particularly exciting, says Schloss. For example, the addition of organic molecules called methyl groups to one of the bases has been shown to play a role in the development of diseases such as cancer. But it is arduous to detect these modifications using conventional sequencing methods, so the full effects and why they happen are still not well understood.

Oxford Nanopore researchers have not yet demonstrated that they can process complete DNA sequences using their system. However, the new results, published this week in Nature Nanotechnology, are an important proof of concept for nanopore sequencing. “They’ve shown the feasibility of all the steps,” says Schloss.

The system that the company used to identify DNA bases is a tunnel-like protein embedded in a membrane very similar to that which surrounds biological cells. The flow of ions across the membrane and through the pore creates a current that can be measured using an electrode similar to those used to study neurons in the lab. By applying a strong electrical potential across the membrane, researchers drive DNA bases through the pore. As each base passes through, it modifies the current flowing across the pore in a characteristic way.

The key to making the method work is controlling the flow of the bases through the protein pore. DNA bases are “too small to be identified on their own: they would fly through,” says James Clarke, a scientist at Oxford Nanopore. So a sugar molecule lining the opening bulks it up so that the DNA doesn’t zip through too rapidly. In previous versions of the nanopore system, this sugar molecule was rather loosely associated with the pore, moving in and out. Company researchers led by founder Hagan Bayley, who is also a professor of chemistry at the University of Oxford, made it possible to read DNA bases one after the other by chemically bonding the sugar to the inside of the nanopore.

Oxford Nanopore can identify bases, but not yet in sequence. The system that it has demonstrated involves passing chopped-up DNA, not whole strands, through the nanopore. The company is now working on a setup for feeding long strands of DNA through the pore one base at a time. To do this, the researchers must attach an enzyme called an exonuclease to the nanopore. They hope that bases will be chopped off one at a time by the enzyme and will pass through the pore to the other side.

“There’s some question [about] what will happen when you put long strands of DNA in front of the nanopore,” says Schloss. “Will it form a hopeless knot?”

This is just one of several unknowns confronting researchers. To make the technology truly scalable and commercially viable, the pores will need to be grouped in large arrays, and the company will need to develop a less complicated way of reading the electrical signals from the pores. Oxford Nanopore says that it is currently working on both of these problems.

A potential pitfall of Oxford’s nanopore-exonuclease approach, says Schloss, is that the DNA will be destroyed as it has been read, making it impossible to resequence a strand to check for errors.

However, there are other approaches to nanopore sequencing that are less destructive. David Deamer, emeritus professor of chemistry at the University of California, Santa Cruz, who first came up with the concept of nanopore sequencing in the 1990s and is a scientific advisor for Oxford Nanopore, points out that this is not the first demonstration of a nanopore system that can identify all DNA bases. Last year, researchers led by Reza Ghadiri at the Scripps Institute, in La Jolla, CA, sequenced a 10-base-long strand of DNA using another nanopore technique. The flow of DNA through the Scripps system, which is based on Deamer’s original concept, is controlled by an enzyme that acts as a ratchet, moving the molecule forward one base at a time. But this system is much too slow, advancing at a rate of one base every 10 minutes, and the Scripps researchers are working on speeding it up.

Oxford Nanopore hasn’t put all its eggs in one basket. It has licensed technology for several nanopore-sequencing methods, including Deamer’s and another that uses an artificial nanopore: a silicon wafer punched with nanoscale holes and lined with carbon nanotubes the conductance of which changes as the DNA passes.

“One of these approaches will have a breakthrough and will be able to sequence at a rate faster and cheaper than what we do now,” predicts Deamer. Neither the researchers at Oxford Nanopore nor those at competing labs are willing to speculate about just when this will happen, or what such a system would cost per genome. But Schloss says it’s possible that one of the groups will meet the National Human Genome Research Institute’s original target year of 2014 for successful nanopore sequencing.

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