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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 (SantaLucia 1998).
This method is less accurate for longer oligos. The formula for the Tm is

Tm=dH/(dS+Rln(Cd/4))-273.15

Where dH is the enthalpy, dS is the entropy, R is 1.987 cal K-1 mol-1, and Cd is the DNA
concentration. dS value is adjusted by 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°C for A and T and
4°C for G and C.

There are several parameters involved in Tm calculation.

DNA concentration is used only in thermodynamic Tm. You may increase oligo DNA concentration
in order to increase Tm.

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.

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.


Formamide concentration affects only the hybridization Tm. You may enter an integer in the
"Formamide (%)" box.

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.

Influencia del Contenido de GC

Oligodesoxirribonucleotido 1
Oligo: 5'-ATATATTCTTATATTTACTATATATATGGGATATATATTA-3'
Primer1: 40 bases
Composition 15 A; 2 C; 3 G; 20 T; 0 OTHER
Percentage: 37.5% A; 5.0% C; 7.5% G; 50.0% T; 0.0%OTHER
MW=12.34 kDa
Hybridization: D:D
Salt: 200 mM
Formamide: 0%
Mismatch: 0 bp
Thermo Tm = 55.5°C Hybridization Tm = 60.0°C GC+AT Tm = 90.0°C

Oligodesoxirribonucleotido 2
Oligo: 5'-ATGATAATGTAGTATGTATATATGTATAGTTGGGGGCCCC-3'
Primer1: 40 bases
Composition 11 A; 4 C; 11 G; 14 T; 0 OTHER
Percentage: 27.5% A; 10.0% C; 27.5% G; 35.0% T; 0.0%OTHER
MW=12.47 kDa
Hybridization: D:D
Salt: 200 mM
Formamide: 0%
Mismatch: 0 bp
Thermo Tm = 68.1°C Hybridization Tm = 70.3°C GC+AT Tm =110.0°C
Oligodesoxirribonucleotido 3
Oligo: 5'-AGGGGAGGGTACCCTAGCATGATATGTAGTTGGGGGCCCC-3'
Primer1: 40 bases
Composition 8 A; 8 C; 16 G; 8 T; 0 OTHER
Percentage: 20.0% A; 20.0% C; 40.0% G; 20.0% T; 0.0%OTHER
MW=12.51 kDa
Hybridization: D:D
Salt: 200 mM
Formamide: 0%
Mismatch: 0 bp
Thermo Tm = 79.1°C Hybridization Tm = 79.5°C GC+AT Tm =128.0°C

Influencia de la variación en la secuencia de nucleótidos

Oligodesoxirribonucleotido 1
Oligo: 5'-ATGGTAACCTGGAGCGCTTGCCAGGACTGCTTTATCGCGA-3'
Primer1: 40 bases
Composition 8 A; 10 C; 12 G; 10 T; 0 OTHER
Percentage: 20.0% A; 25.0% C; 30.0% G; 25.0% T; 0.0%OTHER
MW=12.38 kDa
Hybridization: D:D
Salt: 200 mM
Formamide: 0%
Mismatch: 0 bp
Thermo Tm = 79.1°C Hybridization Tm = 77.4°C GC+AT Tm =124.0°C

Oligodesoxirribonucleotido 2
Oligo: 5'-AATGACCCAGTAGTGCGCGGCTATCTGGTGACTTAGCTGC-3'
Primer1: 40 bases
Composition 8 A; 10 C; 12 G; 10 T; 0 OTHER
Percentage: 20.0% A; 25.0% C; 30.0% G; 25.0% T; 0.0%OTHER
MW=12.38 kDa
Hybridization: D:D
Salt: 200 mM
Formamide: 0%
Mismatch: 0 bp
Thermo Tm = 77.8°C Hybridization Tm = 77.4°C GC+AT Tm =124.0°C

Oligodesoxirribonucleotido 3
Oligo: 5'-CACTCTGAATCTGGTGTATGTAGTGGGCTAGCCACGCGAC-3'
Primer1: 40 bases
Composition 8 A; 10 C; 12 G; 10 T; 0 OTHER
Percentage: 20.0% A; 25.0% C; 30.0% G; 25.0% T; 0.0%OTHER
MW=12.38 kDa
Hybridization: D:D
Salt: 200 mM
Formamide: 0%
Mismatch: 0 bp
Thermo Tm = 76.5°C Hybridization Tm = 77.4°C GC+AT Tm =124.0°C
Influencia de la longitud de la secuencia de nucleótidos

Oligodesoxirribonucleotido 60 Nucleotidos
Oligo: 5'-TAGAATTAATTTATCGTTTATTTTTAACTTCCTCGATGAATAAGGCCTTTTATTGGTTTA-3'
Primer1: 60 bases
Composition 16 A; 7 C; 8 G; 29 T; 0 OTHER
Percentage: 26.7% A; 11.7% C; 13.3% G; 48.3% T; 0.0%OTHER
MW=18.48 kDa
Hybridization: D:D
Salt: 200 mM
Formamide: 0%
Mismatch: 0 bp
Thermo Tm = 71.5°C Hybridization Tm = 70.1°C GC+AT Tm =150.0°C

Oligodesoxirribonucleotido 40 Nucleotidos
Oligo: 5'-TAGAATTAATTTATCGTTTATTTTTAACTTCCTCGATGAA-3'
Primer1: 40 bases
Composition 12 A; 5 C; 4 G; 19 T; 0 OTHER
Percentage: 30.0% A; 12.5% C; 10.0% G; 47.5% T; 0.0%OTHER
MW=12.29 kDa
Hybridization: D:D
Salt: 200 mM
Formamide: 0%
Mismatch: 0 bp
Thermo Tm = 63.6°C Hybridization Tm = 64.1°C GC+AT Tm = 98.0°C

Oligodesoxirribonucleotido 20 Nucleotidos
Oligo: 5'-TAGAATTAATTTATCGTTTA-3'
Primer1: 20 bases
Composition 7 A; 1 C; 2 G; 10 T; 0 OTHER
Percentage: 35.0% A; 5.0% C; 10.0% G; 50.0% T; 0.0%OTHER
MW=6.18 kDa
Hybridization: D:D
Salt: 200 mM
Formamide: 0%
Mismatch: 0 bp
Thermo Tm = 40.2°C Hybridization Tm = 46.0°C GC+AT Tm = 46.0°C

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