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New QProbe System from Japanese Scientists Offers Cost-Effective, Specific qPCR

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Scientists at Waseda University in Tokyo and the National Institute of Advanced Industrial Science and Technology, headquartered in Tsukuba, have developed a new method for performing qPCR that is more flexible, specific, and cost-saving than the current methods on the market. Called the quenching probe, or QProbe, system, their technique takes advantage of the fact that there is a single fluorescent probe at the heart of quantifying the amount of DNA in the sample and it's attached to the probe and not to the DNA itself. Their work was published in July in Analytical Chemistry.

One of the main differences between the QProbe system and conventional qPCR approaches is that the system uses a single fluorescent probe to detect multiple targets. "The same QProbe can be used for any target," says senior author Naohiro Noda. "Thus, this method significantly reduces the cost of [a] real-time PCR setup in comparison with other sequence-specific fluorescent PCR techniques."

The QProbe is a locked nucleic acid oligonucleotide labeled with a single fluorescent molecule. The probe binds to the 3' end of what Noda and colleagues call a universal-tailed nonfluorescent primer, and the entire complex binds to the target to be amplified. Once the QProbe binds, the fluorescent dye is quenched via electron transfer between the dye and a guanine base at a particular position and the QProbe is degraded or displaced by the extension of the reverse primer. The efficiency of the fluorescence quenching is proportional to the amount of target. Using a nonfluorescent 3'-tailed probe ensures that the probe will bind with high specificity. "This method is specific for target sequences," Noda says. "[It] allows the accurate quantification even in the presence of nonspecific PCR products."
Noda and his colleagues used the method to quantify three different model targets, the b-actin, albumin, and b-globin genes, and found that it was comparable to TaqMan and other published methods. They also showed that it could be used to genotype three different SNPs using melting curve analysis.

The new method could be used to improve qPCR for many applications besides genotyping, including medical diagnostics, gene expression analysis, diagnosing infectious diseases, forensics, and the like. Additionally, Noda says, the method could be applied not only to PCR systems but also to other amplification systems such as loop-mediated isothermal amplification, rolling-circle amplification, nucleic acid sequence-based amplification, and helicase-dependent amplification.

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