Quantitative real-time PCR (qPCR) is a widely adopted technique used for scientific, clinical, diagnostic, or quality control purposes. One of the main applications of qPCR is gene expression analysis, although mutation detection, genotyping, DNA detection, and quantification (from pathogens or genetically modified organisms) are also investigated using this technique.Although nonspecific detection based on DNA-binding dyes (including SYBR Green I) offers versatility in qPCR assays, detection of the PCR product using fluorescent probes confers higher specificity and sensitivity to assays, justifying the use of fluorescent probes as a detection method.This chapter seeks to propose a procedure for the design of qPCR assays using fluorescent hydrolysis probe technology. Particular attention will be paid to explaining the steps necessary to ensure the specificity of the oligonucleotides used as primers or fluorescent probes.
Keywords: Fluorophore; Hydrolysis probes; Quencher; qPCR.