SnapGene calculates oligo melting temperature (Tm) values using a nearest neighbor thermodynamic algorithm with up-to-date parameters . This method is the most accurate one available [1,2]. For a given duplex, the calculation accounts for any internal mismatches, loops, and dangling ends.
For a primer binding site, nearest neighbor calculations are combined with dynamic programming  to find the most energetically favorable duplex. Because detailed duplex structures can be distracting, SnapGene shows simplified duplexes by default, but you can see full duplex structures with a setting, see Show Full Structures for Primer-Template Duplexes.
- The Tm depends on the salt concentration. SnapGene assumes a Na+ concentration of 50 mM. This convention is used by many oligo suppliers.
- The Tm also depends on the oligo concentration. SnapGene assumes an oligo concentration of 0.25 μM for a PCR primer, or 0.5 μM each for two annealed oligos.
- For oligos with degenerate (mixed) bases, the Tm is estimated by averaging the relevant parameters.
- If a duplex contains loops, the Tm value is only an approximation.
- Our algorithm employs a two-state melting model, which typically works best for short oligos. Structures such as hairpin loops are not considered. More sophisticated multi-state annealing simulations  are offered by the UNAFold Web Server or the commercial Visual OMP software.
- The monovalent cation concentration in PCR mixtures can vary, and Mg2+ also affects the Tm. Salt-corrected Tm estimates  for a specific buffer can be obtained using the IDT Oligo Analyzer.
- Our algorithm uses accurate parameters  for deoxyinosine (I), and assumes that deoxyuracil (U) will give the same results as thymidine (T), but other non-DNA bases are not supported.
- SantaLucia J Jr, Hicks D. 2004. The thermodynamics of DNA structural motifs. Annu Rev Biophys Biomol Struct 33:415-40. PubMed
- Markham NR, Zuker M. 2008. UNAFold: software for nucleic acid folding and hybridization. Methods Mol Biol 453:3-31. PubMed
- Torgasin S, Zimmermann KH. 2010. Algorithm for thermodynamically based prediction of DNA/DNA cross-hybridisation. Int J Bioinform Res Appl 6:82-97. PubMed
- SantaLucia J Jr. 2007. Physical principles and Visual-OMP software for optimal PCR design. Methods Mol Biol 402:3-34. PubMed
- Owczarzy R et al. 2008. IDT SciTools: a suite for analysis and design of nucleic acid oligomers. Nucleic Acids Res 36:W163-9. PubMed
- Watkins NE Jr, SantaLucia J Jr. 2005. Nearest-neighbor thermodynamics of deoxyinosine pairs in DNA duplexes. Nucleic Acids Res 33:6258-67. PubMed