Faster Genetic Sequencing

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Faster Genetic Sequencing

Postby dignan » Mon Aug 01, 2005 1:22 pm

Anything that speeds up decoding DNA is good news...



DNA Machine May Advance Genetic Sequencing for Patients

August 1, 2005 - NY Times - A new kind of machine for decoding DNA may help bring costs so low that it would be feasible to decode an individual's DNA for medical reasons. The machine, developed by 454 Life Sciences of Branford, Conn., was used to resequence the genome of a small bacterium in four hours, its scientists report in an article published online today by the journal Nature.

In 1995, when the same bacterium was first sequenced, by Claire M. Fraser, it required 24,000 separate operations spread over four to six months, she said in an e-mail message.

The machine uses the chemistry of fireflies to generate a flash of light each time a unit of DNA is correctly analyzed. The flashes from more than a million DNA-containing wells, arrayed on a credit-card-sized plate, are monitored by a light-detecting chip, of the kind used in telescopes to detect the faintest light from distant stars. Then, they are sent to a computer that reconstructs the sequence of the genome.

The decoding or sequencing of genomes has long depended on a chemical process invented by Frederick Sanger in 1977. But genome centers based on this technology are expensive to equip and operate.

For several years biologists have been searching for a method that could be miniaturized and made cheap enough to stimulate a range of new applications.

Among several promising approaches, one called pyrosequencing has been developed by Pal Nyren and Mostafa Ronaghi at the Royal Institute of Technology in Stockholm. The technique depends on separating the double helix of DNA into single strands, and building up new strands to complement the old ones. As each new DNA base is added to a growing strand, a chemical component known as pyrophosphate is discarded.

Dr. Nyren's team developed the chemistry to convert the discarded pyrophosphate into a trigger for luciferase, the enzyme that fireflies use to generate their light. Because the chemists knew in each cycle which of the four DNA bases they had added, a flash of light indicated the sequence at that point.

Dr. Ronaghi, who is now at Stanford University, said that he and Dr. Nyren had developed the chemistry and showed it could be miniaturized, and that 454 Life Sciences, having licensed their patents, had made the system practical.

"What they have done here is very significant," Dr. Ronaghi said, noting that the company had already sequenced 50 microbial genomes. "This is the first step toward $1,000 human genome sequencing," he said.

The Joint Genome Institute, a federal genome sequencing center in Walnut Creek, Calif., has ordered one of 454's $500,000 sequencing machines but has not yet installed it. Paul Richardson, the institute's head of technology development, said the new approach "looks very, very promising" and could reduce sequencing costs fourfold.

The machine's limitation is that at present it can only read DNA fragments 100 units or so in length, compared with the 800-unit read length now attained by the Sanger-based machines. The shorter read length makes it harder to reassemble all the fragments into a complete genome, Dr. Richardson said, so although microbial genomes can be assembled with the new method, mammalian genomes may be beyond its reach at present.

Dr. Fraser, director of the Institute for Genomic Research in Rockville, Md., also said that the new machine's short read lengths "limit its overall utility at this point."

Jonathan Rothberg, board chairman of 454 Life Sciences, said the company was already able to decode DNA 400 units at a time in test machines. It was working toward sequencing a human genome for $100,000, and if costs could be further reduced to $20,000 the sequencing of individual genomes would be medically worthwhile, Dr. Rothberg said.

There would be little advantage at present in sequencing a patient's entire genome, but in the medicine of the future, complete documentation about an individual's genetic makeup could well provide prognosis or indicate a preferred treatment.

The new technology avoids a pitfall of the Sanger method, which is that the fragments of DNA to be analyzed are first amplified by being cloned in bacteria. But the bacteria cannot handle certain fragments, leaving gaps in the genome sequence. In the new technique, each fragment of DNA is captured in an individual drop of liquid and amplified to 10 million copies with a well-established chemical method known as the polymerase chain reaction.

The 10 million copies from each droplet are then attached to an ultra-small bead, and the beads are dropped into a credit-card-sized grid of 1.6 million wells, where the pyrosequencing takes place. Each time the correct base is added to the fragments of DNA on a specific bead, a flash of 10,000 photons is picked up through the bottom of the wells by the light-detecting chip that sits under the small grid of wells. A computer can reconstruct the sequence of bases composing the fragments stuck to each bead, and from the overlaps between fragments can reassemble the entire genome from which they were derived.

http://www.nytimes.com/2005/08/01/healt ... oo&emc=rss
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dignan
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