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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