Thermostable Ligase Temperature Calculator

Nearest-neighbour Tm calculation

The nearest-neighbour model predicts DNA duplex stability by summing thermodynamic contributions of each overlapping dinucleotide pair (e.g. 5′-AG-3′ stacked on 3′-TC-5′). The SantaLucia (1998) unified parameters provide ΔH and ΔS for all 10 unique Watson–Crick dinucleotides.

Where ΔH is in cal/mol, R = 1.987 cal/(mol·K), and Ct is the total strand concentration in molar. This gives the Tm at 1 M NaCl; the salt correction adjusts ΔS for the actual buffer ionic strength.

Salt correction

Monovalent cations stabilise DNA duplexes by neutralising the phosphate backbone charge. At lower salt concentrations, electrostatic repulsion destabilises the duplex and lowers Tm. The SantaLucia (1998) correction:

This adds a per-base-pair entropy term proportional to ln[Na⁺]. At 1 M NaCl (ln = 0), the correction vanishes. Typical ligase buffers contain 25 mM KOAc or KCl, giving a significant reduction in Tm compared to the 1 M standard.

Thermostable ligase reaction design

In a ligase detection reaction (LDR) or oligonucleotide ligation assay (OLA), two probes hybridise adjacently on a target template. The ligase seals the nick only when there is perfect complementarity at the junction. Temperature must be:

1. High enough to melt mismatched probe–template duplexes (discrimination) 2. Low enough that perfectly matched probes stay hybridised (sensitivity)

The sweet spot is typically 5–10 °C below the lower probe Tm. Probes should be designed with similar Tm values and a Tm gap $\geq$ 5 °C above the mismatch Tm for reliable single-base discrimination.