Sequence utilities / Primer Tm and properties
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Tm_NN is the nearest-neighbour thermodynamic Tm (the recommended reference); Tm_Wallace (4·GC + 2·AT) is a rough estimate for 14–20 nt primers only; Tm_GC is the empirical GC formula. Degenerate primers are expanded into every specific sequence: Tm_NN is their mean and the next column gives the lowest–highest value (number of variants in brackets).
Tm depends on reaction conditions: enter the actual Na⁺/K⁺, Mg²⁺, dNTP and primer concentrations. With Mg²⁺ the sodium equivalent follows von Ahsen 2001; typical PCR uses 1.5–2.5 mM Mg²⁺ and 200–500 nM primer, giving a Tm a few degrees above the default conditions.
The two primers of a pair should ideally differ by no more than 2–3 °C; the pair check treats consecutive rows as a pair (forward then reverse).
Self-complementarity, 3′-end complementarity and hairpin flags are quick base-pairing hints, not secondary-structure free energies; consider redesigning primers with several flags.
Nearest-neighbour parameters: SantaLucia 1998 (PNAS 95:1460); salt corrections: SantaLucia 1998, Owczarzy et al. 2004 (Biochemistry 43:3537), von Ahsen et al. 2001 (Clin Chem 47:1956); Wallace rule: Thein & Wallace 1986. Results match Biopython MeltingTemp.Tm_NN (DNA_NN3).
Up to 500 primers; primer lengths of 10–60 nt are recommended.
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