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Rice U Scientists Develop Technology to Optimize Hybridization Capture Yield in Multiplex Settings


NEW YORK (GenomeWeb) – Scientists from Rice University have demonstrated a new method to adjust, on the fly, the capture yield of multiple targets in a single experiment. They published their results earlier this month in Nature Methods.

"It's a probe technology that's broadly applicable to many other technologies," David Zhang, senior author of the paper, told GenomeWeb. It's based on previous work Zhang has done creating competitive probes that bind to similar nucleic acid sequences to enrich the target sequence.

Because of inherent variation in probe binding efficiency, imbalances arise when trying to get different probes to bind multiple targets in the same reaction. Zhang said this technology fixes those imbalances by spiking the reaction with probes that draw down the concentration of one target while keeping another constant. Whether it's in multiplex PCR, microarray analysis, or next-generation sequencing enrichment, "any technology where you're trying to look at multiple species at once could benefit from this rebalancing," he said.

Zhang and his co-authors call this "continuous tuning" and are hoping to make sequencing or hybridization panel enrichment kits to be sold by his startup firm, Searna.

Previously, researchers didn't have a way of individually adjusting the amounts of just one kind of target in a sample, Zhang said. In a hybridization step, they could change the temperature or buffer conditions of the experiment, but that leads to either an increase or decrease of all targets. "It's not a decoupled way of making more of one" target, Zhang said. 

Getting more equivalent capture efficiency could be particularly attractive in NGS applications, where some reads are more prevalent than others because they can be captured with a low yield. "After the PCR step, there is always some amount of bias," for one target over another, Zhang said, which leads to inefficient sequencing and a bottleneck effect.

By using probes to take the more efficiently hybridized targets out of the equation, the numbers even out and aren't as biased towards one or more targets. "We're allowing the same number of reads to be more efficient," Zhang said. "That potentially allows you to pool more, different patient samples together. You don't need as many reads to get the same amount of information."

Zhang has plans for Searna to commercialize this technology as a kit for enriching panels of genes. The firm would design kits for specific panels using knowledge about the capture efficiencies to rebalance the final concentrations of each target before analysis.

Though the firm is focused on fundraising and developing the technology, and hasn't yet started any collaborations on clinically relevant applications, it has filed for patents on the method, he said.

Zhang has high hopes that he and the company will be rewarded on their patent claims. He recently was awarded a patent on early research into what he calls a toehold probe, which leverages the thermodynamics of nucleic acid binding to optimize hybridization. That application was filed in 2011.

"We're pretty optimistic" that the new applications will be recognized, even if it takes a similar amount of time, he said. "We think these kinds of things are going to be able to stand up under scrutiny and be issued."

In the meantime, Searna is trying to validate the technology, especially the X-probe technology with which the new tuning method works in concert, using commercially available samples.

The firm is already gaining steam as it searches for funding and a permanent location from which to conduct business. Grace Lu, Searna's CEO and co-founder, said that the firm has secured lab space at the Johnson & Johnson Innovation Labs in South San Francisco, California. "We're welcome to move in there when we have money," she said.

Searna has also added Harvard Medical School's George Church and Dan Marshak, formerly of Perkin Elmer, to its advisory board.

Searna is in the process of fundraising and has also applied for a Small Business Innovation Research Grant from the National Institutes of Health and the National Cancer Institute.