NEW YORK (GenomeWeb) – A new design for an oligonucleotide probe shows the potential to be able to combine PCR amplification and microarray hybridization into a single step for molecular diagnostics. The technology, called Structured Cleavage Induced Single-Stranded Oligonucleotide Hybridization Reaction (SCISSOHR) probes, could pave the way towards the manufacture of universal microarrays.
Led by Laurie Girard and Michel Bergeron, scientists at Université Laval in Quebec City designed strands of DNA shaped like a hairpin, consisting of three main segments. The first is a DNA probe that is complementary to the target for PCR amplification. Connected to this is a loop that is also complementary to the target, forms the hairpin turn, and connects the PCR probe to a complex that includes a segment designed to hybridize to the microarray capture probe. This microarray arm itself has a loop, formed by the sequence complementary to the microarray capture probe, joined on either side by sequences that are complementary and match up to the PCR DNA probe, so that hydrogen bonds keep the oligonucleotide in a hairpin formation.
At the head of the structure, a fluorophore is attached to the end of the microarray segment and a quencher is attached to the end of PCR probe, in proximity to the dye. If the target DNA strand is present, polymerase will open up the hairpin and sever the SCISSOHR, releasing the microarray-bound segment and separating the fluorophore from the quencher. This leaves the sequence to hybridize to the microarray capture probe and be detected optically. Even if the target is not present and the probe hybridizes to the microarray, the quencher will stay in proximity to the fluorophore.
"Every part counts," said Maurice Boissinot, a professor of microbiology at Université Laval and a co-author of a paper describing the technology published in November in Analyst.
The SCISSOHR structures might in the future allow for PCR and microarray integration. "If you can design a universal microarray, you just have to change the part of the probe to be tailored to your target and you have the universal part that could be hybridized to the capture sequence," Boissinot said. This could greatly simplify manufacturing the microarrays, since they would all be the same.
The probes overcome a major restriction of microarrays, which is that some sequences are not amenable to capture probes and it's impossible to predict which sequences can't be hybridized, he said. And because the hybridizing sequence can be the same for almost every SCISSOHR probe, it eliminates the need to validate a new microarray for every DNA target.
Though it shows promise, the technology is currently unrefined. "This is a proof-of-concept study," said Karel Boissinot, a co-author on the paper and coincidentally a distant cousin of Maurice. The equipment used in the study was jury-rigged from supplies left over from a previous study. Karel Boissinot said that to test the SCISSOHR probes, they dropped a microarray chip dropped into a PCR tube and borrowed optical equipment to read the microarrays. "We want to go further into developing applications and improving technology so it could be much more attractive for other scientists," he said.
Maurice Boissinot said that the university is applying for a patent and that the team is already in talks with potential industry partners who are interested in the technology, though he declined to name them. He thinks that SCISSOHR probes could be useful in detecting microbes and their drug-resistant genes, screening for genetic mutations, and for cancer diagnostics and other personalized medicine applications.
"At the moment, we want to improve the platform by having a dedicated platform and a dedicated container," Maurice Boissinot said. "From that we could work out the library [of hybridization sequences]. Once we have that, we will be in better shape to determine if we want to start our own company or approach a potential licensee."