How does SnapGene estimate oligonucleotide (primer) melting temperatures (Tm)?

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 in SnapGene Preferences.

Assumptions

  1. The Tm depends on the salt concentration. SnapGene assumes a Na+ concentration of 50 mM. This convention is used by many oligo suppliers.
  2. 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.
  3. For oligos with degenerate (mixed) bases, the Tm is estimated by averaging the relevant parameters.

Limitations

  1. If a duplex contains loops, the Tm value is only an approximation.
  2. 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.
  3. 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.
  4. 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

  1. SantaLucia J Jr, Hicks D. 2004. The thermodynamics of DNA structural motifs. Annu Rev Biophys Biomol Struct 33:415-40. PubMed
  2. Markham NR, Zuker M. 2008. UNAFold: software for nucleic acid folding and hybridization. Methods Mol Biol 453:3-31. PubMed
  3. Torgasin S, Zimmermann KH. 2010. Algorithm for thermodynamically based prediction of DNA/DNA cross-hybridisation. Int J Bioinform Res Appl 6:82-97. PubMed
  4. SantaLucia J Jr. 2007. Physical principles and Visual-OMP software for optimal PCR design. Methods Mol Biol 402:3-34. PubMed
  5. Owczarzy R et al. 2008. IDT SciTools: a suite for analysis and design of nucleic acid oligomers. Nucleic Acids Res 36:W163-9. PubMed
  6. Watkins NE Jr, SantaLucia J Jr. 2005. Nearest-neighbor thermodynamics of deoxyinosine pairs in DNA duplexes. Nucleic Acids Res 33:6258-67. PubMed
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