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ZS Genetics' Chemistry Allows EM to See DNA; Sequencing Product Targeted for 2008

Wouldn’t it be great if one could simply place a strand of DNA under a microscope and read off its bases? ZS Genetics, a Massachusetts-based startup company, is trying to do just that.
The company has been working on a single-molecule long-read method to sequence DNA by electron microscopy and plans to have its first commercial sequencer ready by mid-2008 after targeting gene expression analysis later this year. Earlier this month, ZS Genetics presented its technology and commercialization plans publicly for the first time at the New Hampshire MIT Enterprise Forum in Manchester, NH.
“[The reason] why no one else has done this is that DNA is invisible under an electron microscope,” because it lacks heavy atoms that create the necessary contrast, William Glover, the company’s founder and president, told In Sequence.
To overcome this, ZS Genetics has invented a PCR-based method that converts normal DNA into DNA that can be “seen” under the EM, using proprietary labeled nucleotides in the PCR reaction. These nucleotides contain atoms with high atomic numbers to create contrast.
Researchers then plan to attach long pieces of the modified DNA — up to 20,000 base pairs in length — to a substrate and stretch the DNA out by applying a liquid flow.
After drying the substrate, they image the DNA using a “modified” transmission electron microscope with sub-angstrom resolution, Glover said. The bases, which can be distinguished by their shape, he said, are read out using automated pattern recognition software.
So far, the scientists have replaced two of the four nucleotides with the labeled versions, but in order to sequence DNA, “we need at least three labels, and we would like to have four,” Glover said.
Right now, they can visualize several thousand bases of DNA at a time “but because we don’t have enough distinct labels, we cannot really tell what the sequence is,” Glover said.
Also, since polymerases do not incorporate enough different modified nucleotides efficiently, “in order to have three or four good labels, we will probably need to evolve a polymerase,” he added.
The company potentially also faces a number of other technical challenges: One is preventing several thousand bases of DNA from becoming tangled, “which seems perhaps the most daunting aspect,” commented David Stokes, a professor of structural biology at New York University School of Medicine and an electron microscopy expert, in an e-mail message.
Glover claims the liquid flow technique, details of which he declined to provide, “effectively separates the strands, which flow over one another quite smoothly.”
Also, reading the labeled bases from long stretches of DNA will not be trivial, Stokes said. “A gene might have 3,000 bases, which would be 1 micrometer if stretched out. In order to see the [bases with the required] resolution, you need to be at high magnification and so you will have to drive the microscope along the length of the DNA and take a series of images,” he wrote. “This will require sophisticated coordination between imaging software and microscope control.” Also, he added, “all this tracking and imaging will take a long time.”
According to Glover, each image, which will take one second to acquire, will cover 3,000 bases.
ZS Genetics holds three published US patent applications on the process, all entitled “Systems and methods of analyzing nucleic acid polymers and related components.”
While it is working on its sequencing technology, the company is preparing to bring its first commercial product to market, a gene expression analysis system based on the same technology. In order to measure gene expression, though, the researchers only need to count labeled cDNA molecules instead of sequencing them and thus do not need more different labels.
By the middle of this year, the company hopes to offer custom-made oligo-based microarrays for electron microscopy that will each have approximately 3,000 user-defined features, each half a micrometer in diameter.
By mid-2008, ZS Genetics is planning to launch its sequencing product, which will be targeted to resequencing human genomes in whole or in part.

“[The reason] why no one else has done this is that DNA is invisible under an electron microscope.”

But the high up-front cost of the electron microscope might deter users from acquiring it. “The technology has a high capital cost, so it’s really suited for people who want to do many genomes rather than a few,” Glover said.
An electron microscope with sufficient resolution in the sub-angstrom range costs on the order of $5 million, according to Glover. However, sequencing an entire human genome with the technology will cost $5,000-$10,000 in consumables and labor, he estimated.
Glover, who has an undergraduate degree in chemical engineering from California State University, Long Beach, and has worked previously in commercial nuclear power and management consulting, founded ZS Genetics in 2005 after developing his idea for the system for two years prior to this. “ZS” stands for “zero science.”
 “What we mean by that is, we are using technologies that are proven and mature,” said Tom Abert, the company’s vice president of finance and its first angel investor. “What’s different is how we put them together, and the specific way in which we use them.”
The company has seven employees at the moment and no lab space of its own. Instead, it works with approximately 15 contractors at universities, small private laboratories, and instrument vendors, according to Glover. “We have done most of our lab work at local universities and a local company that specializes in electron microscopes,” he said.
Glover declined to mention any of the local partners by name, citing bilateral non-disclosure agreements, but Japanese EM manufacturer JEOL has its US headquarters in Peabody, Mass., and is mentioned in ZS Genetics’ patent applications. ZG Genetics lists North Reading, Mass., as its address in the applications. Also, a publicly available database document from the University of New Hampshire shows that ZS Genetics sponsored a $24,000 collaboration with Kelley Thomas, a professor for genomics and co-director of the university’s Hubbard Center for Genome Studies, last year, entitled “Phase I Optimization.”
So far, the company has raised $1.8 million from angel investors. It is currently working on a $500,000 equipment lease through a local bank, and is negotiating with an undisclosed electron microscope vendor for a $5 million equipment contribution. In addition, ZS is planning to raise another $3.5 million in equity later this year for strategic investments.
The company does “not expect to require capital after that point,” Glover said. “We are planning on selling the company rather than trying to build the business ourselves, and we are very open to chatting with folks who are interested in a business relationship.”

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