Table 1. Selection criteria for designing comparative microsatellite primers.
| Repeats | Stability of primer secondary structures (ΔG*) | |||||||||||
| Lexpected | Lprimer † | Tm ‡ | ΔTm | %GC§ | 2-6× | 1× | 3′ HP | Int HP | 3′ SD | Int SD | 3′ CD | Int CD |
| <350 | 18–22 | 58–62 | <1 | 45–60 | <3 | <6 | >−2.00 | >−3.00 | >−5.00 | >−6.00 | >−5.00 | >−6.00 |
Lexpected: expected length of PCR products (bp); Lprimer: primer length (bp); Tm: melting temperature (°C); ΔTm: Tm difference between both primers; %GC: G+C content; 2-6×: number of tandemly repeated non-mononucleotide motifs (2–6 bp); 1×: length of mononucleotide runs; ΔG: Gibbs free energy required to break the secondary structure (kcal/mol); 3′: 3′-end of primers; Int: Internal; HP: hairpin, SD: self-dimer, CD: cross-dimer.
*
Output from NetPrimer; criteria as recommended in the application's manual.
†
Exceptionally up to 26 bp.
‡
Output from PrimaClade.
§
A 30–62% range was tolerated for primers >22 bp.