TaqManprober

TaqManprober, commonly referred to as a TaqMan probe, is a hydrolysis-based DNA probe used in real-time PCR to quantify a specific sequence during amplification. The technology is widely used in diagnostics and gene expression studies. It was developed by Applied Biosystems (now Thermo Fisher Scientific) in the 1990s and became a foundational component of the TaqMan assay family.

A TaqMan probe is a short oligonucleotide complementary to a target region between PCR primers. It carries a fluorescent reporter dye at the 5' end and a quencher at the 3' end. During PCR, the 5' to 3' exonuclease activity of Taq polymerase cleaves the probe, separating the reporter from the quencher and increasing fluorescence in real time. The signal correlates with target quantity and can be monitored cycle by cycle. Multiplexing is possible by using probes with distinct fluorescent reporters to monitor multiple targets simultaneously.

Applications include quantitative DNA measurement, gene expression analysis, pathogen detection, and genotyping using allele-specific TaqMan probes. The method offers high specificity and a closed-tube workflow, reducing contamination risk relative to endpoint assays. Multiplexing capabilities enable simultaneous surveillance of several targets in one reaction.

Design considerations include selecting a probe within the amplicon, appropriate length and melting temperature compatible with primers, and balanced reporter-quencher chemistry. Variants such as minor groove binder (MGB) probes can improve specificity, and the use of dark quenchers can reduce background fluorescence. Proper controls, standard curves, and assay validation are recommended to ensure accurate quantification.