It is well appreciated that Mg2+ stabilizes duplex DNA 80 to 100 fold to as much as 140-fold (1). How ever Promega (2) and other online calculators of primer Tm do not take into account the effects of magnesium on helix stability. I personally ran a multiplex PCR which its annealing temperature is close to Tm calculated without magnesium effect. I used Wetmore and Sninsky (1995) N-N formula with a little change in [salt] calculation, [salt]= [K+] + [NH4].

 

Now my question is, do we need to consider magnesium effect for Tm calculation of primers in PCR? since doing so raises Tm about 5-10 oC?

 

[PLEASE SUPPORT YOUR ANSWERS WITH PUBLISHED ARTICLES IF POSSIBLE]

 

References

(1) Nakano S, Fujimoto M, Hara H, Sugimoto N. Nucleic acid duplex stability: influence of base composition on cation effects. Nucleic Acids Res 1999;27:2957–65.

(2) http://www.promega.com/biomath/calc11.htm#salt_Desc

(3) Wetmur, J.G., and J.J. Sninsky (1995). Nucleic acid hybridization and unconven- tional bases. In PCR Strategies (M.A. Innis, D.H. Gelfand, and J.J. Sninsky, eds.). Acade mic Press, San Diego, CA, pp. 69–83.

It really depends. For practical purposes approximations without salts are often used. Overall, the empirical approach tends to give results more reliably. For more accurate assessments, nearest-neighbor thermodynamic theories with corrections for salts are used. There are oodles of tools that include it (I think primer3 does it, for example). There was work from Honda in the 90s or from Schütz that dealt with that. I would look for the terms and the authors as a starting point.