TaqMan

TaqMan is a real-time polymerase chain reaction (qPCR) technology developed by Applied Biosystems (now part of Thermo Fisher Scientific) that uses sequence-specific, dual-labeled hydrolysis probes to quantify nucleic acids. In a typical TaqMan assay, a probe carrying a fluorescent reporter dye at the 5' end and a quencher at the 3' end binds to the target DNA between the forward and reverse primers. During PCR amplification, the DNA polymerase’s 5' to 3' exonuclease activity cleaves the probe, separating the reporter from the quencher and producing an increase in fluorescence that correlates with product accumulation. Fluorescence is measured in real time, allowing determination of cycle threshold (Ct) values for quantification.

Compared with non-specific DNA-binding dyes like SYBR Green, TaqMan probes add an additional layer of specificity because the fluorescence signal requires probe binding to a complementary target sequence. The approach also supports multiplexing, as different targets can be detected simultaneously by using probes labeled with distinct fluorophores. TaqMan assays are commonly supplied as pre-designed, validated primer–probe sets for particular genes or variants.

Applications of TaqMan include gene expression analysis, SNP genotyping, pathogen detection, and copy number variation assessment. The method is widely used in clinical diagnostics and research due to its specificity, sensitivity, and the straightforward interpretation of results. Limitations include higher cost from probe synthesis and the need for careful probe design to accommodate sequence variation or mutations in the target region.