Tm (Melting Temperature)熔解温度
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Melting temperature
Choosing the Primer | Melting Temperature command opens the Melting Temperature dialog box. The oligo sequence in memory is displayed in the Oligo sequence box. DNAMAN calculates the length, GC% and the molecular weight of the primer according to the sequence and displays the results in the dialog box.
The melting temperature of an oligonucleotide is calculated with three methods:
1.Thermodynamic Tm:
The Tm is calculated using the nearest-neighbor thermodynamic values method (Breslauer et al). This method is less accurate for longer oligos. The formula for the Tm is
Tm=dH/(dS-Rln(Cd))-273.15+16.6*(log10(Cs))
Where dH is the enthalpy, dS is the entropy, R is 1.987 cal K-1 mol-1, Cd is the DNA concentration, and Cs is the salt concentration.
2.Hybridisation Tm:
This method is generally used for DNA or RNA hybridization, especially in presence of high salt and formamide. It is more accurate with longer oligos. The Tm is calculated using the following formula:
for DNA:DNA hybridization:
Tm=81.5+16.6*(log10[Na+])+0.41*[%(G+C)]-0.63*(%Formamide)-500/L-1.5(%Mismatch) for DNA:RNA hybridization:
Tm=79.8+18.5*(log10[Na+])+0.58*[%(G+C)]+11.8*[%(G+C)]2-0.5*(%Formamide)-820/L-1.5(
%Mismatch);
for RNA:RNA hybridization:
Tm=79.8+18.5*(log10[Na+])+0.58*[%(G+C)]+11.8*[%(G+C)]2-0.35*(%Formamide)-820/L-1.5(
%Mismatch);
* where L is the length (bases) of the oligonucleotide.
3.GC+AT Tm:
The estimated Tm is the sum of the contribution of each base: 2℃for A and T and 4℃for G and C.
There are several parameters involved in Tm calculation.
1.DNA concentration is used only in thermodynamic Tm. You may increase oligo DNA
concentration in order to increase Tm.
2.Salt concentration affects thermodynamic Tm and hybridization Tm. In the [Na+][mM] box,
you may type a suitable salt concentration of your experiments. The value must be an integer greater than 0.
3.Hybridization type is required for Hybridization Tm. In the DNA/RNA box, enter the
hybridization type:
D:D for DNA and DNA hybridization
D:R for DNA and RNA hybridization
R:R for RNA and RNA hybridization.
4.Formamide concentration affects only the hybridization Tm. You may enter an integer in the
"Formamide (%)" box.
5.Mismatch number also affects only the hybridization Tm. You may type in the "Mismatch"
box a mismatch number of base pairs between the oligo and DNA.
With all parameters entered, click the Show Tm button to display the melting temperatures. You may change the sequence in the Oligo sequence box and then click the Show Tm button to recalculate the length, GC%, MW and Tm of the oligo.
Click the Report button to display the analysis results in a text window. You may save it as a text document.
Example:
Oligo:
5'-GACTGCCACTTCCTCGA TGAAGG-3'
SEQ Primer 1: 23 bp;
Composition: 5 A; 7 C; 6 G; 5 T; 0 OTHER
Percentage: 21% A; 30% C; 26% G; 21% T; 0% OTHER
Molecular Weight (kDa): ssDNA: 7.06
Hybridization type: DNA:DNA
Salt concentration: 50 mM
Formamide: 0 percent
Mismatch: 0 bp
Thermo Tm = 60.6 ℃%GC Tm = 57.0 ℃GC+A T Tm = 72.0 ℃。