July/August 2008

Sequencing a Single Molecule of DNA

Continued from page 1

By Emily Singer

When a scientist activates the machine, a precisely choreographed dance of fluids begins. An enzyme called DNA polymerase and a single type of fluorescently labeled base--say, A--flow into the cell. The enzyme causes those As to take their places in growing strands of DNA that complement the strands in the samples. (Each of the four bases can pair with only one other base, so an added A must line up opposite an existing T, and a C against a G.) Once the fluorescently tagged base is incorporated into the new strand, the HeliScope's camera can spot the light it emits. "The imager detects a plume--a 200-­nanometer cone of light--from the integration of a single [base] onto a ­single strand of DNA," says Steve Lombardi, president of Helicos.

Other advanced sequencing methods use a similar approach, known as sequencing by synthesis. But unlike those technologies, the HeliScope can distinguish the unamplified fluorescent signal of a single base taking its place on a growing DNA strand. One key to that ability is a nonstick material that the company developed, which coats the surface of the flow cell and allows it to be washed clean between reactions: residual fluorescent bases would make it more difficult to accurately detect individual sequencing reactions. "You need to make sure no extra base molecules are sticking to the surface," says Patrice Milos, chief scientific officer at Helicos. "This was one of the biggest early challenges." After each cycle, the fluorescent markers are clipped from the newly incorporated bases, and remaining chemicals are washed away. The process is repeated sequentially with each of the four bases.

The HeliScope generates a massive amount of raw data every second. It takes five to ten days to read all the DNA that can be loaded into two flow cells; for sequencing, that's 400 million strands of DNA per cell, which can generate 20 billion bases' worth of usable sequence. Scientists load the machine, press a button on its face, and leave. But the sequencing obsessed can use the Internet to check the machine's progress in the middle of the night, a common occurrence at Helicos.

Once the HeliScope creates its series of fluorescence photographs, an accompanying data-processing center converts them into strings of letters. Software pastes these pieces together to form a longer sequence.

Missing Mutations
In a paper published in Science earlier this year, scientists reported on their use of the HeliScope to sequence the genome of the M13 virus, important proof that single­-­mole­cule sequencing could be used to read and ­assemble the sequence of a complete genome. (Approximately 7,000 base pairs long, the M13 virus's genome is tiny--about a millionth the size of a human's.) The technology is so new that it's not yet clear what applications it will be best suited to. But some scientists believe that single-­molecule sequencing could be particularly important in understanding how genetic variations contribute to disease. After all, some rare mutations linked to disease may have been missed in previous genomic studies because they weren't copied during the amplification process.

Helicos is still tinkering with the tech­nology, developing chemistry that could boost the speed of the sequencing reactions and allow more pieces of DNA to be anchored to a flow cell. Along with the other major players in the field, the company hopes to deliver a complete genome sequence for $1,000, an accomplishment that would mark the beginning of something totally new in medicine: individuals' ability to access their own genomic information.

Emily Singer is TR's biotechnology and life sciences editor.