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Life Technologies Sees Bright Future for Quantum Dots in G3 Sequencer


Quantum dots will play an important role in Life Technologies' third-generation, or G3, sequencing platform, according to a company official.

The company is currently developing the single-molecule platform, which it plans to test with external customers starting next year (see In Sequence 2/10/2009), as it builds on the experience of various parts of its business in different technology areas. One "early-access collaborative technology partner" will be the J. Craig Venter Institute, according to a company spokesperson.

"We are now getting quite successful putting [the pieces] together to be a first research-phase embodiment of this technology," Andy Watson, vice president of business operations for advanced genomic systems at Life Technologies, told In Sequence during a visit to the company's headquarters in Carlsbad, Calif., last week.

He mentioned that company researchers are currently working with "different synthetic templates" but are not yet showing sequencing results because they want to ensure they live up to people's expectations for the technology. "We will see how some of the things come along in the lab right now. We will publish and present when we are ready for it," he said.

The new sequencer will use fluorescence energy resonance transfer, or FRET, to detect the incorporation of nucleotides into a growing DNA strand. In FRET, energy is transferred from one dye molecule to another when they come into close proximity.

Quantum dots will play an important role in this process, according to Watson, a co-founder of Quantum Dot, a company that Invitrogen acquired in the fall of 2005 for an undisclosed sum. He joined Invitrogen about a year ago to head the commercial development of the third-generation sequencer.

Quantum dots, or "Qdots," are nanometer-sized semiconductor crystals that emit bright light in a range of sharp colors. Two favorable properties, compared to small organic fluorescent dyes, are their photostability and their brightness.

After Invitrogen consolidated Quantum Dot into its Molecular Probes business in Eugene, Ore., which it had acquired in 2003, company scientists started working internally on combining quantum dots and organic dyes for FRET, according to Watson. They published a paper in 2006 in Analytical Biochemistry, though he stressed that this was not the first time a research group had used quantum dots for FRET.

Over the past several years, Invitrogen also became interested in the DNA-sequencing space, where it had limited experience at the time, and soon realized that quantum dots and FRET-based single molecule detection could potentially be harnessed for sequencing.

As the company worked on "demonstrating feasibility" for FRET-based sequencing, and "built out some instrumentation capability" internally, Watson said, it also became interested in VisiGen Biotechnologies, which had been working independently on using FRET for DNA sequencing (see In Sequence 5/8/2007)

Invitrogen acquired VisiGen for $20 million last fall, both for its technology expertise and for its intellectual property (see In Sequence 10/28/2008), and maintains VisiGen's Houston team and site, according to Watson.

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Applied Biosystems, which merged with Invitrogen last November, brought a few more pieces to the table, including expertise in building instrumentation; informatics and software capabilities; and "some of the chemistry work" conducted at its Foster City, Calif., site.

FRET Sequencing

For Life Tech's version of FRET sequencing, researchers attach a quantum dot near the polymerase, primer, and template complex. When nucleotide triphosphates — dye-labeled at their terminal phosphate group — get incorporated, their labels become "energized" by the quantum dot and emit light before they are cleaved off and diffuse away.

In analogy to Pacific Biosciences, which limits the observation volume by creating an evanescent field in a zero-mode waveguide, in FRET sequencing, only fluorescent dyes in close proximity to the quantum dot are being observed.

"[The system] can have lots and lots of dye molecules floating around, but they look invisible, they don't get energized because they are not close enough to the quantum dot," Watson explained.

Quantum dots help with the system in two ways, he said: When the dye molecule on the nucleotide starts emitting light, the quantum dot signal decreases at the same time, so two signals can be measured for the same incorporation event. The reverse happens when the dye molecule starts to leave.

"This concept of correlated signals, we think, is going to be quite important in terms of being able to make sense of these signals that come from looking at single molecules, because they are quite noisy," according to Watson.

Secondly, quantum dots can help funnel large amounts of excitation light to the fluorescent dye molecules, acting "almost like a collector" of light. "We believe this will remove the need for very high-powered laser types of excitation systems," Watson said.

Though the company has not yet determined any product specifications for the G3 sequencer, Life Tech sees it "fitting in a very complementary way to the SOLiD system."

Starting in 2010, the company plans to test the new platform with external partners, including the J. Craig Venter Institute. Life Tech is co-funding a new, two-year sailing expedition by JCVI and intends to use its new sequencing technology to analyze water samples collected during the trip. "We look forward to partnering with Craig [Venter] on this journey and on using new innovative technologies, such as single-molecule DNA sequencing, to decipher the genetic code of these new microbial species," said Greg Lucier, the company's chairman and CEO, in a statement last week (see Short Reads in this issue).

However, for the next two to three years, the company believes the SOLiD system will outperform any third-generation real-time sequencer in terms of throughput, cost per base, and raw accuracy, Watson said — largely because of the density of the information and the intensity of the signal it acquires.

Third-generation sequencers, on the other hand, are likely to have a leg up on data-turnaround time as well as read length. And although Life Tech has not yet determined the configurations for its G3 sequencer, it could potentially be better suited than SOLiD or other second-generation sequencer for running smaller numbers of samples and not require barcoding.

Watson said it is too early to try and compare Life Tech's system to PacBio, which has not yet announced the commercial specs for its single-molecule sequencer, but has said it plans to ship its first commercial instruments in the second half of 2010 (see In Sequence 2/10/2009).

"We will see how they actually perform in the market; we will see what their price points are," Watson said.

Overall, he said, "we don't see the G3 system as being the next one in a linear fashion" that will replace existing first- and second-generation sequencers. "Sure, the G3 is going to cannibalize a little bit each of those, but I think mainly it's going to [create] its own market area."