What are the differences in parameter settings between the various modes within the RealTimeDesign™ software?
The 'Less Restrictive' parameter set relaxes the guidelines on amplicon lengths (up to 150 bases) and a longer probe length (28 bases). The parameters demanding a close proximity between the forward primer and probe are also extended slightly to 12 bases.
The 'Least Restrictive' parameter set permits an amplicon length of up to 200 bases, a long probe design (30 bases), and probes that may anneal up to 20 bases distant from the forward primer.
All three parameter sets adhere to the dogma of dual-labeled probe design. Assays proposed to any one of the three are expected to work well.