Designing a good qPCR assay requires careful consideration of a number of parameters that can spell the difference between success and failure, according to Mark Behlke, M.D., Ph.D., CSO, Integrated DNA Technologies (IDT). “The design of the assay must carefully determine parameters such as primer placement, specificity, avoidance of single nucleotide polymorphisms, oligonucleotide interactions, and accurate melting-temperature calculations.”
One answer is the use of predesigned assays that shift the burden of these choices to the manufacturer. But not all assays are created equal, according to Dr. Behlke. “Unfortunately, databases are dynamic, not static. Some commercial assays were designed using now-outdated databases. In other cases, a scientist might be using a primer design format that was done with older, less accurate melting-temperature algorithms. Even worse, a researcher may not be able to find out from the company the sequence information that could be verified or even a correct quote for a research publication.”
To overcome this problem, IDT offers its PrimeTime® predesigned qPCR assays. Scott D. Rose, Ph.D., director, molecular genetics, reports, “We’ve developed these assays for all genes in the human, mouse, and rat transcriptomes. The assays are very specific and eliminate concerns such as cross-reactivity within each genome and primer interactions. Importantly, we also freely provide all the information a researcher will need to know, including full sequence disclosure of both primers and probes as recommended by the MIQE guidelines.”
The IDT technology employs its ZEN double-quenched probes. Dr. Rose indicated that “these assays provide sensitivity down to 10 copies or less. We were able to reduce background fluorescence by decreasing the length between the reporter dye and the internal quencher to only nine base pairs. Traditionally probes have 20–30 bases between the dye and quencher.
“Thus, the ZEN quencher is always within close proximity of the reporter, resulting in a much lower initial background fluorescence, Lower background results in greater sensitivity for the assay. With the ZEN system, even long probes—up to 50 base pairs—are effectively quenched because the distance between the quencher and the reporter dye is fixed and is independent of probe length.”