The molecular beacons were designed according to the guidelines available at http://www.molecular-beacons.org. They consisted of probe sequences 2425 nucleotides long, 6-nucleotide arm sequences, the fluorophore FAM covalently linked to the 5' end, and the quencher DABCYL linked to the 3' end. The melting temperatures of the hairpin stems and probe sequences were 6366C. To estimate the stability of the molecular beacons hairpin stem structure, the DNA folding program m-fold (available at http://www.bioinfo.math.rpi.edu/~mfold/dna/form1.cgi) was utilized. Selected primer and probe sequences for this study are shown in Table 2.
Real-time PCR analysis was performed using the iCycler iQ detection system. The conditions of the reaction were carefully optimized by varying molecular beacon (50250 nM), primer (2001,000 nM), and Mg2+ (35 mM) concentrations and the annealing temperature (5560C). The optim