Peter Mouritzen has designs on LNA. Credit: 360DEGREES

The conventional wisdom on probe-based real-time PCR assays says that if you have 10,000 genes to detect you will need 10,000 different probes to detect the PCR products. And to provide the required specificity, the probes need to be at least 18 nucleotides long. A team of researchers and bioinformaticians at Exiqon in Vedbaek, Denmark, is challenging this dogma. By scanning the genomes of key species they have come up with a set of 90 locked nucleic acid (LNA) 8- and 9-mer probes that they claim can be used to identify every gene on which you will ever work. Because of the greater rigidity of LNAs, even a single mismatch with the potential target will seriously affect binding and prevent generation of signal, thus giving the same specificity as a longer regular oligonucleotide probe.

The probes come to life when used with Exiqon's software, which for each target gene designs an optimum primer pair in combination with a specific probe. “The short LNA probes enable the technology, but the software is the key,” says Exiqon's new technology-development manager, Peter Mouritzen. To identify the most specific combination, the software performs what Exiqon calls in silico PCR. This checks primer–probe predictions against the entire genome and transcriptome of the organism, minimizing the risk of possible mispriming which could produce a false-positive signal from a real-time PCR assay.

For RT-PCR you want to look at the transcribed sequence and not the genomic DNA, which could contaminate the sample, so the software increases specificity by choosing a section where the primers span an exon–exon splice junction. While the primer could hit the genomic DNA, it will not generate a signal, because the intron is likely to make the product so long that it will not be amplified efficiently.

Users buy the LNA probes from Exiqon but can get their primers from any supplier. The assay design software is free online. “Normally people spend hours designing a PCR assay, and this thing does it in seconds,” comments Mouritzen.

Roy Bicknell at the Weatherall Institute of Molecular Medicine at the University of Oxford, UK, agrees. “We look for new genes expressed on tumour vasculature as anticancer targets, so when we identify them by bioinformatics we have to do a lot of validation. Because we are looking at many genes we need to keep making new primers — and that is the beauty of the Exiqon system — it's for people who want to look at lots of different genes,” he says.