Molecular beacon probes form stem-loop hairpin structures at low temperature. In this closed state, the fluorophore and the quencher are held in close vicinity, and thus no fluorescence signals are detected (Tyagi and Kramer, 1996). The hairpin loop contains gene specific sequence for hybridization in real-time PCR. Thus, the rules for TaqMan® probe design also apply to molecular beacon loop design. The hairpin structure is disrupted by hybridization to the amplicon, which separates the fluorophore from the quencher for fluorescence detection. Because of this unique structural requirement, one major task for molecular beacon probe design is to identify a suitable hairpin structure that melts 7-10°C higher than the PCR primers. The Tm of the hairpin stem cannot be calculated using the Tm formulas for PCR primers because the stem is formed by intramolecular folding. In general, programs for secondary structure prediction, such as Mfold (Zuker, 2003), can be used to predict hairpin stem melting temperature. The stem usually consists of 5-7 base pairs with 75-100% GC content. A G residue should not be placed at the end of the stem because it will quench the fluorophore.
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