As a qPCR trainer, I often get the question why to worry about careful design and validation of dye-based assays when an easier alternative is available: TaqMan probes. This question results from the widespread believe that probe-based assays are superior in specificity to assays that rely on SYBR Green I chemistry for detection.

In this blog post, I want to nuance this point of view: dye-based assays are perfect for many real-time qPCR applications. When you understand the true advantages and disadvantages of probe-based assays, you can reduce the cost while keeping the quality of your qPCR experiments high. And, in the end, isn’t that what we all want?

Designing a well working and reliable qPCR assay is a lot of work. Apart from prediction of specificity, the assay should also be screened for possible secondary DNA structures that interfere with efficient amplification (see Figure 1 in ) and for the presence of single nucleotide polymorphisms (SNPs) that impair amplification of the variant allele.