So, you have a primer sequence and you want to know what its melting temperature is? Well, MacVector has a lot of Primer Design functionality, and sometimes its difficult to know where to start. If you have MacVector 12.6 or later, the easiest and quickest way to do this is using the Quicktest Primer functionality. There are two main ways to use this;
(1) Choose Analyze | Primers | Quicktest Primer to open the Quicktest Primer dialog, then paste or type your primer sequence into the main edit box.
(2) Select a primer-sized region (10 to 80 residues) of a sequence in an existing MacVector sequence window and choose Analyze | Primers | Quicktest Primer. This will open the Quicktest Primer window and fill the edit box with the selected sequence.
Either way, the Quicktest Primer Dialog will open;
There are three Tm values reported (circled in red) – which one to use? In general, for primers under ~35 residues in length, we recommend you use the “Santa Lucia” Tm. Unfortunately, calculating the predicted Tm of a primer is not an exact science. The only real way of determining the Tm is to perform a melting temperature experiment under the conditions you intend to use the primer. You can control those conditions from settings available by clicking on the Parameters button. Over the years, computer algorithms have advanced such that the calculated estimates now more closely match the observed experimental data. I don’t want to get too much into the hairy details of Tm prediction algorithms, but here’s the rationale for the MacVector results, in reverse order;
Baldino – this algorithm is preferable for long primers (35nt or greater). It was first described by Bolton and McCarthy (1962) and slightly modified by Baldino et al (1989). It is based purely on the length and G+C% of the primer and the concentration of monovalent cations in the reaction.
Breslauer – this algorithm uses more accurate nearest neighbor thermodynamic calculations using the equations described in Rychlik et al. (1990) with the thermodynamic parameters of Breslauer (1986). This is the calculation used in MacVector prior to version 12.6 and is left in primarily for backwards compatibility.
SantaLucia – this is a similar algorithm to the Breslauer algorithm except that it uses the updated combined thermodynamic parameters of SantaLucia (1998). This is widely regarded as one of the most accurate Tm calculation algorithms currently available and is the one we recommend to use, particularly for primers shorter than 35 nt.
Both the Breslauer and SantaLucia algorithms also now take into account the effects of divalent cations (e.g. Mg++) and the concentration of deoxynucleotides in the reaction mix as described by von Ahsen et al. (2001). This means that if you do want to confirm the Tm of a primer as it was calculated in versions of MacVector prior to 12.6, you should click on the Parameters button and set the Divalent cation conc and Nucleotide conc values to “0”, then observe the Breslauer Tm value. However, normally we would recommend you set those values to the actual concentrations you use experimentally.
To summarize – for typical primers of less than 35 nucleotides, use the SantaLucia Tm and make sure the divalent cation and nucleotide concentration parameters are set to the values you use experimentally. Primer3 uses the same calculations internally and will produce identical results. For longer primers, you may prefer the Baldino Tm calculation. The Breslauer Tm is only reported for backwards compatibility with MacVector 12.5.
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