By Ben Butkus
Boreal Genomics has developed a prototype sequence- and methylation-enrichment and –detection platform that can enrich target DNA sequences 10,000-fold compared to background DNA that differs by as little as a single nucleotide, the company said recently.
Coupled with Aurora, the company's beta-stage electrophoresis-based platform for DNA concentration and purification, as well as a proprietary detection technology still under development, Boreal believes that the sequence-enrichment technology could push the company beyond the sample-prep market and into clinical point-of-care diagnostics, Andre Marziali, Boreal's president and CSO, told PCR Insider this week.
Founded in 2007, Vancouver, BC-based Boreal has for the past several years been prepping its flagship platform, Aurora, for commercialization. Aurora is based on a technology called synchronous coefficient of drag alteration, or SCODA, which uses rotating electric fields to selectively concentrate nucleic acids in an agarose "focusing" gel based on physical characteristics such as charge species, linear charge density, size, and conformational entropy.
Boreal has demonstrated that Aurora can generally separate nucleic acids from difficult contaminants such as particulate matter and PCR inhibitors with 100-fold higher yield and purity than commonly used technologies such as elution columns.
As such, the company has been beta-testing Aurora with various undisclosed laboratories interested in using the instrument to purify DNA from difficult and complex environmental samples, such as those commonly encountered in forensics analysis or even archaeology.
The platform also holds promise in purifying nucleic acids from complex clinical samples such as blood or stool, and Boreal has been eyeing those markets, as well.
"We're doing collaborations [for Aurora] in the clinical area, and our own development in that area," Marziali said. "But the beta testing is mostly around difficult environmental samples, which obviously includes forensics."
Aurora is in the final stages of beta-testing and now likely "a couple of months" from commercialization, Marziali said. But even as Boreal preps Aurora for market, it has turned its attention to the "second and third tiers" for the SCODA technology: the prototype sequence- and methylation-enrichment platform and the early-stage, sequence-specific detection technology.
Like the Aurora platform, Boreal's sequence-enrichment technology uses SCODA. However, the key addition of an acrylamide gel containing short nucleic acid probes allows the SCODA technology to purify the DNA through a sequence-specific filter of sorts.
"The thing about acrylamide that makes it useful for this application is that we can attach DNA to it," Marziali said. "We decorate the entire gel with short 20-mers with a particular sequence, and the interaction between those probes in the gel and the target DNA that gets injected is what drives the focusing force in that case."
The concept is almost the opposite of capture hybridization enrichment, Marziali added. "We run the gel at the melting temperature between the probe-target duplex, and that leads to non-linearity in the motion of the target that drives focusing in the way the Aurora system does," he said. "So a lot of the technology in the sequence-enrichment system is similar to the Aurora, but the gel sort of provides a sequence-dependent friction, if you will, to the fragments."
Earlier this month at the Knowledge Foundation's Sample Prep 2011 conference in San Diego, Marziali presented preliminary data from in-house studies demonstrating the technology's ability to enrich a particular sequence that differs from as many as 10,000 copies of background sequences by a single base pair.
"Right now we're working with researchers who are interested in detecting very rare DNA sequences in a population of other DNA," Marziali said. "This is ultimately where the technology's strength is going to be."
In some ways, he said, the new technique competes with highly-sensitive iterations of PCR; and in some way, it complements them, Marziali said.
"For enrichment of low-abundance targets, in a sense it could compete with something like digital PCR, but ultimately it can also be used as a front end to digital PCR," he said. For instance, if a researcher could use digital PCR to find single mutant sequences present in 106 copies of background sequences, "we could add another four orders of magnitude on top of that. So if you needed to find something that was present in part in 1010 background DNA, you can imagine using our enrichment followed by digital PCR."
But competitively speaking, "one of the nice things about our system is because we're not doing PCR — not creating any new genetic material — there is no possibility that you will create a sequence that wasn't there in the first place," Marziali said. "All we do is enrich. PCR can, through substitution errors, create sequences that weren't present in the original population, and in fact amplify them to create noise."
Boreal also has evidence that its technology can enrich for methylated DNA, even down to identical sequences that differ by a single methylation.
"That enrichment is more like 100-fold … because it is such a fine separation in binding energy to the probe," he said. "Work remains to be done there, but we believe we can separate identical sequences that differ only by methylation pattern."
Such sensitivity makes the new technology promising for clinical applications, which would be a new realm for Boreal.
"I think we have an enormous opportunity in clinical diagnostics in looking for rare sequences in clinical samples that might be tumor markers or other disease markers or pathogens that are present in a high background of human DNA," Marziali said.
In order to explore such applications, the nucleic acid purification and enrichment technologies would need to be coupled with sequence-specific detection technology, which Boreal is also developing.
At the Sample Prep 2011 conference, Marziali described this technology as a "phase-sensitive algorithm for highly sensitive, sequence-based optical detection." This week, he further characterized the detection technology as taking advantage of certain inherent features of the sequence-enrichment technology.
"In a way, it would use the sequence-enrichment technology to perform the specificity of the detection," Marziali said. "It would basically localize your target sequence in a region … [and then] you don't have to distinguish it from other sequences."
In contrast, real-time PCR, for instance, achieves specificity by detecting a certain sequence of interest against a background of sequence using a sequence-specific probe; and achieves sensitivity through amplification of the target sequence.
"We're splitting those things up over two technologies," he said. "So the sequence enrichment provides specificity; and then the detection scheme provides sensitivity. Once we've isolated a very specific sequence to a region of the gel, then the challenge simply becomes detecting it with enough sensitivity."
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