The length and the GC content of the stem sequence is designed in such a way that at the annealing temperature of the PCR, and in the absence of the target, the molecular beacons remain closed and non-fluorescent. In practice, the length of the probe sequence usually falls in the range between 15 and 30 nucleotides.Īfter selecting the probe sequence, two complementary arm sequences are added on either side of the probe sequence. The prediction should be made for the probe sequence alone before choosing the stem sequences.
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The melting temperature of the probe-target hybrid can be predicted using the 'percent-GC' rule or 'nearest neighbor' rules (available in most probe or primer design software packages).
If, on the other hand, single-nucleotide allele discrimination is not desired, longer and more stable probes can be chosen. In order to discriminate between amplicons that differ from one another by as little as a single nucleotide substitution, the length of the probe sequence should be such that it dissociates from its target at temperatures 7-10 ˚C higher than the annealing temperature of the PCR.
The probe sequence of the molecular beacon should be so long that at the annealing temperature of the PCR it is able to bind to its target. If you are designing molecular beacons to detect the synthesis of products during polymerase chain reactions, you can select any region within the amplicon that is outside the primer binding sites. The process of molecular beacon design begins with the selection of the probe sequence.
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The fluorescence versus temperature profiles of the molecular beacon that were used in this example indicate that the molecular beacon is suitable for assays that are performed below 55 ˚C, because below 55 ˚C the free molecular beacons remain dark, yet the probe-target hybrids form spontaneously and are stable. It is important to note that the probe-target hybrid melting temperature can be adjusted independently from the melting temperature of the stem by selecting a target region of appropriate length. The longer the probe and the higher its GC content, the higher the melting temperature of the probe-target hybrid. The temperature at which the probe-target hybrid melts apart depends upon the GC content and the length of the probe sequence. At low temperatures, the probe-target hybrid remains brightly fluorescent, but as the temperature is raised the probe dissociates from the target and tends to return to its hairpin state, diminishing the fluorescence significantly. How the fluorescence of the probe-target hybrid varies with the temperature is indicated by the red fluorescence versus temperature trace. If a target is added to a solution containing a molecular beacon at temperatures below the melting temperature of its stem, the molecular beacon spontaneously binds to its target, dissociating the stem, and turning on its fluorescence. The temperature at which the stem melts depends upon the GC content and the length of the stem sequence. However, at high temperatures the helical order of the stem gives way to a random-coil configuration, separating the fluorophore from the quencher and restoring fluorescence. As shown by the green fluorescence versus temperature trace below, at lower temperatures molecular beacons exist in a closed state, the fluorophore and the quencher are held in close proximity to each other by the hairpin stem, and there is no fluorescence.
In order to design molecular beacons that function optimally under a given set of assay conditions, it is important to understand how their fluorescence changes with temperature in the presence and in the absence of their targets.
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