If the alternative structures are a result of the choice of the stem sequence, the stem sequence can be altered and if the alternative structures arise from the probe sequence itself, the frame of the probe can be slided along the target to obtain probe sequences that are not self-complementary. As with PCR primers in general, the sequence of the molecular beacon should be checked against those of the primers using a primer designing software program to make sure there are no areas of complementarity which may cause the molecular beacon to bind to primers and increase background. The primers used during molecular beacons PCR experiments should be designed to produce a relatively short amplicon, in general less than 150 bp. The molecular beacon is an internal probe which must compete with the opposite strand of the amplicon for binding to its complementary target. Having a shorter amplicon allows the molecular beacon to compete more efficiently for binding to its target and, therefore, produces better results during molecular beacon PCR experiments.
Molecular beacons are very versatile.
The general design, outlined above, can be altered to fit specific applications. For instance, provided that the molecular beacon will be long enough to have a Tm enabling it to bind to its target at the PCR annealing temperature, the shorter the loop sequence, the better able the molecular beacon is to discriminate single base pair mismatches in templates. On the other hand, in experiments in which it is desirable for one molecular beacon to report the presence of multiple targets differing by a single base pair, the loop sequence should be longer so that the Tm's of mismatched targets are above the PCR annealing temperature. Multicolor molecular beacons for allele discrimination. In situ visualization of mRNA for basic fibroblast growth factor in living cells.