RPA & MIRA Primer Rules
Established parameters and guidelines for designing highly efficient primers for isothermal amplification assays.
Length & Size
Optimally 30 to 36 nt. Shorter reduces kinetics; longer increases secondary structures. (Exception: highly sensitive 22 nt reverse primers).
Short amplicons outcompete noise. Ideal range: 100 - 200 bp (acceptable up to 500 bp).
Composition & Tm
Target 40 - 60%
Target 35 - 60%
Unlike PCR, Tm is not critical. Some guidelines suggest 50 - 100 °C.
Terminal Ends
Terminate with G or C.
CRUCIAL: Avoid complementarity at the 3' ends to prevent primer-dimers.
Avoid placing multiple 'G' nucleotides within the first 3 to 5 base pairs.
Repeats & Structures
Avoid long continuous tracks. Max mononucleotide repeat (e.g., CCCCC) is 5.
Avoid targeting regions with direct/inverted repeats and palindromes.
Discard sequences promoting secondary structures or hairpins.
Tolerances & Concentrations
System is highly robust! It tolerates 5 to 9 bp mismatches without affecting performance. Keep degenerate bases to a minimum.
150 - 600 nM
750 - 2000 nM
Empirical Screening Strategy
Because performance cannot be reliably predicted purely by sequence, standard practice dictates designing 8 to 10 forward & reverse candidates and screening them empirically in a matrix.