Myeloid Leukemia

Real-Time RT-PCR Strategies 37
2.1.2. Specific Fragment Detection
In order to add further specificity to quantitative PCR, specific fragment
detection must be employed. Six variant chemistries are reported.
2.1.2.1. TAQMAN PROBES OR HYDROLYSIS PROBES
The TaqMan assay (Perkin-Elmer–Applied Biosystems) is characterized by
using three oligonucleotides (two template-specific primers and one probe) and
a DNA polymerase with 5� exo-nuclease activity (15). Taq and Tht polymerase
are the two polymerases used most commonly.
The probe is characterized by a fluorophore that is covalently attached to
one end and by a quencher that is covalently attached to the other end (Fig. 6).
Upon excitation, the reporter passes its energy, via fluorescent resonance energy
transfer (FRET; or, Förster-type energy transfer), to the quencher while
the probe is intact (16). The principle is that when a high-energy dye is in close
proximity to a low-energy dye, energy transfers from high to low. The quencher
reduces the fluorescence of the fluorophore when the two moieties are separated
by