Thursday, September 25, 2008

Faster, Cheaper DNA Sequencing

A U.K. company hopes to dramatically reduce the cost of reading a person's genome.

By Neil Savage

The race to build a fast, inexpensive device to sequence individual human genomes has a new entrant: a British company that uses a technique called nanopore sequencing. Cheaper genome sequencing--the fabled $1,000 genome--could lead not only to medical treatments tailored to a patient's specific genetic makeup, but also to better ways to diagnose disease, identify biological threats, and discover new drugs.

The company, Oxford Nanopore Technologies, has raised approximately $20 million in funding and recently licensed separate nanopore technologies developed at the University of Oxford, Harvard University, and the University of California, Santa Cruz.

Like the sequencing systems from Helicos BioSciences of Cambridge, MA, and Pacific Biosciences of Menlo Park, CA, nanopore sequencing can analyze individual strands of DNA, eliminating much of the time and expense of sample preparation. In a conventional DNA-sequencing scheme, the DNA sample must be copied many times.

But nanopore sequencing has the added advantage of doing away with the fluorescent molecules typically used to label DNA bases so that their sequence can be read by cameras. "We think we're the only label-free technology out there," says company president Gordon Sanghera. "There's no labeling. There's no amplification. And that really does take out the bulk of what you're doing." He says that sample preparation for the nanopore system takes hours, rather than the days it takes for others.

Nano sequencer: An exonuclease (in green) slices the DNA into individual bases, which pass through the blue nanopore and bind to the pink cytodextrin, giving off characteristic electrical signals. The nanopore system is built into an array on a silicon chip.
Credit: Oxford Nanopore Technologies

Nanopore sequencing is not as mature as other technologies, says Jeffrey Schloss, program director for technology development at the U.S. National Human Genome Research Institute (NHGRI). "The challenge is that people haven't read DNA sequences yet with nanopores," he says. But he adds that "we seem to be getting very, very close with Oxford Nanopore."

Nanopores are naturally occurring proteins about a nanometer in diameter that provide openings in cell membranes so that the cells can eject or ingest materials. (To appreciate how tiny nanopores are, consider that, the average human hair is about 100,000 nanometers thick.) The Oxford system employs an alpha-hemolysin protein produced by the Staphylococcus bacterium, which uses it to extract the contents of other organisms' cells. The Oxford team places the nanopore in an artificial membrane made of lipids. Above and below the membrane are a solution of salt and a pair of electrodes. An electrical current can flow from the solution above to the solution below, but only through the nanopore, because the membrane has a high electrical resistance.

Inside the nanopore is a cytodextrin, a sugar molecule. A single strand of DNA is small enough to slip through the nanopore, and as it passes through, each of the four nucleic acids binds briefly to the cytodextrin, then lets go again. When an acid binds to the sugar, it disrupts the flow of the electrical current in a recognizable way, so telling the base cytosine from guanine, or adenine from thymine, is a simple matter of reading the characteristic electrical signal.

In the researchers' first conception of the system, the entire strand of DNA would pass through as a whole. In theory, bases could bind to the sugar at a rate of a million per second, but that's much too fast to generate a discernible signal. So Hagan Bayley, Oxford Nanopore's founder and an Oxford University chemical biologist who developed some of the technology the company has licensed, added another component to the system: an exonuclease, an enzyme that slices the DNA strand into individual bases and directs them through the nanopore. Each base passes through the nanopore individually and sequentially, slowing the overall rate down to between 10 and 50 bases every second, giving the electrical equipment time to read the signal, which is in the range of a picoamp, a trillionth of an amp.

The company will speed up the analysis of DNA by putting an array of these nanopore setups on a silicon chip. Sanghera says they'll start with several hundred nanopores per chip, and then eventually expand to tens of thousands.

Though the company has demonstrated the individual pieces of the system, it still has to put them all together. It's working on ways to attach the exonuclease to the protein and trying to marry the biological part to the sensing electronics and the silicon chip. The company doesn't have to invent any new electronics, but it does need to design custom chips, which can be complex. "The challenge is system integration, and it's a big challenge," Sanghera says.

Though he won't reveal a specific launch date, he says the company is planning to keep pace with Pacific Biosciences, which aims to have a sequencing device on the market in about two years.

The researchers are aiming for the $1,000 genome. The hope is that by bringing the price of sequencing down to that range, individuals could afford to have their genomes recorded, allowing doctors to tailor treatments to their particular genetic makeup. Sanghera sees the next generation of nanopore technology replacing the biological nanopores with artificial ones. Ultimately, the goal is to make the system solid-state, built wholly in silicon the way computer chips are.

NHGRI has long championed cheap gene sequencing. It has given more than $12 million to the researchers whose technology Oxford Nanopore licensed, including a recent $6.5 million grant to Daniel Branton and Jene Golovchenko of Harvard. The institute's initial goal was to achieve the $1,000 genome by 2014. "I think that's turning out to be a pretty good estimate," Schloss says.