This command will calculate varied melting temperatures for sequences between 8 and 100 nucleotides long and send the result to the Output window.

1. Choose Analysis > Melting Temperature in the main menu or Sequence Analysis > Melting Temperature in the selection contextual menu:

or click on the Melting Temperature button in the Sequence toolbar:

2. Open the Output multitab window to access the Tms.

Basic Tm

This method approximates the Tm for sequences < 14 nucleotides with the formula:

Tm= (wA+xT) * 2 + (yG+zC) * 4

(Marmur,J., and Doty,P. (1962) J Mol Biol 5:109-118)

and for longer sequences the following equation is used:

Tm= 64.9 +41*(yG+zC-16.4)/(wA+xT+yG+zC)

(Wallace,R.B., Shaffer,J., Murphy,R.F., Bonner,J., Hirose,T., and Itakura,K. (1979) Nucleic Acids Res 6:3543-3557).

This method is for comparison only and is not recommended for usage at the bench.

Salt-Adjusted Tm

This method calculates a salt-adjusted Tm for sequences < 14 nucleotides with the equation:

Tm= (wA+xT)*2 + (yG+zC)*4 - 16.6*log10(0.050) + 16.6*log10([Na+])

and for longer sequences the following equation is used:

Tm= 100.5 + (41 * (yG+zC)/(wA+xT+yG+zC)) - (820/(wA+xT+yG+zC)) + 16.6*log10([Na+])

This is the recommended method for long oligonucleotides. By default, GenBeans uses a primer concentration of 250 pM and [Na+] of 50 mM.

Nearest-Neighbor Tm

This method calculates a Tm using the nearest-neighbor method according to SantaLucia J.Jr (1998). Proc. Natl. Acad. Sci. USA 95:1460-1465. This is the preferred method for sequences between 8 and 40 nucleotides.

Tm PCR

For PCRs, GenBeans determines the best method to calculate the melting temperature depending on the length of the primers at a standard salt concentration of 50 mM and standard oligonucleotide concentration of 250 pM. Practically GenBeans uses the nearest-neihbor method for primer shorter than 40 nucleotides and the GC-content method for longer oligonucleotides:

Tm = 81.5°C + 16.6°C x log[Na+K] + 0.41°Cx(%GC) - 675/N

where N is the length of the sequence. This formula has been reported more accurate for long oligonucleotides than the salt-adjusted method.