Table 1 Primer design selection criteria. These criteria are used, where applicable, for determining the compatibility of forward and reverse primers within a given locus and for pair-wise compatibility between primers for different loci.
From: Computational tradeoffs in multiplex PCR assay design for SNP genotyping
Parameter | Allowed Range |
|---|---|
Length | 17 – 24 bases |
% GC | 35 – 65% |
Tm (nearest neighbor) | 57.0 – 70.0 C |
Tm Difference | 3.0 C (for both candidate primer pairs and for all primers within a particular multiplex tube) |
Base repeats | ≤ 3 bases maximum |
Product Size | 60 – 200 bases |
Distance to SNP | 177 bases (5' end) 5 bases (3' end) |
Self complementarity local alignment score | ≤ 8.0 (match = 1.0, mismatch = 1.0, gap = -2.0) |
3'-Tail alignment ΔG | ≥ -4.5 kCal/mol |