The 'Most Restrictive' parameter set dictates very stringent guidelines for assay design. The resulting assays propose a short amplicon length (up to 100 bases), a short probe design (25 bases), a short interval between the forward primer and probe (no more than 10 bases distant) and no stable misalignments within and between the primers. While a robust PCR reaction may tolerate rule breaking without performance impact, this parameter set errs on the conservative side of caution. We recommend attempting to design your assay using the 'Most Restrictive' parameter set whenever possible.

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.