Help for Haiti

Pacific Biosciences and Harvard scientists decode genome of Haitian cholera pathogen

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MENLO PARK, Calif.—Scientists from Pacific Biosciences of California Inc. and Harvard Medical School have successfully employed single-molecule, real-time (SMRT) DNA sequencing technology to rapidly characterize the pathogen responsible for the recent deadly cholera epidemic in Haiti.

The research points to a South Asian origin of the Haitian outbreak and may lead to new preventive or therapeutic strategies. Published online last month in the New England Journal of Medicine, the results provide the first whole-genome sequence analysis and most detailed genetic profile to date of the Haitian Vibrio cholerae strain and confirm that the cholera pathogen now present in Haiti is closely related to the "El Tor O1" variant from South Asia, previously unknown in the Caribbean or Latin America.
In this collaboration, DNA prepared from five V. cholerae strains at Harvard Medical School was received by Pacific Biosciences in November.

"Through the truly remarkable and dedicated efforts of Dr. Eric Schadt and his colleagues at PacBio, we had a good understanding of the genome of the Haitian V. cholerae isolates and their likely origin by Friday evening, Nov. 12," states Dr. John Mekalanos, chair of the department of microbiology and molecular genetics at Harvard Medical School, and a senior author on the study. "This understanding has important public health policy implications for preventing cholera outbreaks in the future."
One of the fundamental challenges with observing a DNA polymerase working in real time is the ability to detect the incorporation of a single nucleotide, taken from a large pool of potential nucleotides, during DNA synthesis.

"To resolve this problem, we applied the same principle that operates in the metallic screen of a microwave oven door," says Dr. Eric Schadt, chief scientific officer at PacBio and co-author of the paper.
He points out that in a microwave oven, the screen is perforated with holes that are much smaller than the wavelength of the microwaves. Because of their relative size, the holes prevent the much longer microwaves from passing through and penetrating the glass. However, the much smaller wavelength visible light is able to pass through the holes in the screen, allowing food to be visible.

"We have reduced this same principle to the nanoscale and we call our innovation a zero-mode waveguide, or ZMW," he states.

Started by a group of physicists at Cornell, PacBio moved to the San Francisco Bay area to access both the technical expertise and the venture capital concentrated in the region and in seven years developed the system used to sequence the Haitian cholera strain in a mere two days.

"And much of that time was spent fixing things that broke," says Schadt. 

In the PacBio process, the DNA polymerase is immobilized on the floor of the ZMW. Phospholinked nucleotides are introduced into the SMRT Cell from above. As the nucleotides diffuse through the bottom 30nm of each ZMW in the SMRT Cell, a small noise signal is generated. When the DNA polymerase encounters the nucleotide complementary to the next base in the template, it is incorporated into the growing DNA chain. During incorporation, the DNA polymerase holds the nucleotide for tens of milliseconds, orders of magnitude longer than the average diffusing nucleotide. While held by the polymerase, the fluorescent label emits colored light. The PacBio RS— a platform for single-molecule, real-time detection of biological events—detects this as a flash whose color corresponds to the base identity, which is recorded. Following incorporation, the signal immediately returns to baseline and the process repeats, with the DNA polymerase continuing to incorporate multiple bases per second.

"Now armed with a more complete characterization of this pathogen, the scientific community is empowered with information that can be used to inform public health policy decisions such as the appropriate use of vaccines to quell this epidemic," says Schadt. "The ability to quickly and easily perform real-time monitoring of pathogens also opens the door to using this technology as a routine surveillance method for public health protection in addition to pandemic prevention and response."

PacBio plans to release its first production SMRT machines during the first half of 2011 to usher in an era of "molecular epidemiology," as Schadt puts it. The company already numbers about 450 staffers and projects eventual sales for its SMRT technology to reach billions of dollars.

In 2009, the sequencing market was estimated to be $1.2 billion, about evenly split between first and second generation sequencing and is expected to grow to more than $3.6 billion by 2014 according to Scientia Advisors, a market research firm.

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