SnapGene calculates oligo melting temperature (Tm) values using a nearest neighbor thermodynamic algorithm with up-to-date parameters [1]. 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 [3] 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.
Assumptions
- 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.
Limitations
- 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 [4] 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 [5] for a specific buffer can be obtained using the IDT Oligo Analyzer.
- Our algorithm uses accurate parameters [6] for deoxyinosine (I), and assumes that deoxyuracil (U) will give the same results as thymidine (T), but other non-DNA bases are not supported.
References
- 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