nearest neighbour tm prediction
nearest neighbor oligo tm calculation
PCR primer design
oligo melting temperature
oligo tm calculator
alternative to oligoanalyzer
primer dimer analysis
predict primer dimers
PCR Primer Inspector@molbiotools.com
A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics
SantaLucia J. Jr.
Proc Natl Acad Sci U S A. 1998;95(4):1460-5.
Oligonucleotide melting temperatures under PCR conditions: nearest-neighbor corrections for Mg(2+), deoxynucleotide triphosphate, and dimethyl sulfoxide concentrations with comparison to alternative empirical formulas
von Ahsen N., Wittwer C. T., Schütz E.
Clin Chem. 2001;47(11):1956-61.
Predicting stability of DNA duplexes in solutions containing magnesium and monovalent cations
Owczarzy R., Moreira B. G., You Y., Behlke M. A., Walder J. A.
The thermodynamics of DNA structural motifs
SantaLucia J. Jr., Hicks D.
Annu Rev Biophys Biomol Struct. 2004;33:415-40.
As the dissociation equilibrium and hence the melting temperature depends on the total concentration of the annealing oligonucleotides, such information has to be included in the Tm predictions.
Cations stabilize DNA duplexes by diminishing repulsive forces between negatively charged phosphate groups of nucleotides. The total concentration of monovalent cations is expectedly a sum of concentrations of sodium, potassium and ammonium cations (not all of them have to be present in a PCR reaction mix). It is also advised to add the concentration of the dissociated fraction of Tris in the PCR reaction mix (a half of the total Tris concentration may be a good approximation). The Tm prediction may be less accurate in high concentrations (above 100 mM) of monovalent ions capable to form hydrogen bonds (ammonium, Tris) and for bulkier monovalent ions (tetramethylammonium).
Magnesium cations strongly affect DNA duplex stability so their concentration must be provided in order to estimate the melting temperature accurately.
FREE DEOXYNUCLEOTIDE TRIPHOSPHATES
The effect of dNTPs on primer annealing is mediated by their strong binding of magnesium cations in 1:1 molar ratio. As magnesium cations affect the DNA duplex stability substantially, the dNTPs concentration must be reflected in any serious melting temperature calculation.
MELTING TEMPERATURE CALCULATION PURPOSE
The melting temperature is slightly different in a situation when the primer is present in large molar excess to the PCR template and in a situation when two complementary oligonucleotides anneal in an approx. equimolar solution.
MELTING TEMPERATURE CALCULATION
All the melting temperature calculations provided here are based on the nearest neighbor method with parameters described in references  and . Results of two different algorithms (references  and ) for monovalent and magnesium cation correction are offered.
PRIMER DIMER PREDICTION
The primer dimer stability prediction is based on the nearest neighbor method with parameters described in the reference . The stability score is the opposite value of a predicted ΔG37°C, 1M NaCl (kcal/mol). This value should only be seen as an approximate relative measure of duplex stability as accuracy of the prediction cannot be guaranteed.
Some dinucleotide sequences containing one mismatch have been found to contribute to duplex stability with ΔG values similar to regular Watson-Crick A/T pairs . Such mismatches are marked by broken vertical bars in the alignments. Colons mark unfavorable pairs, i.e. those whose pairing would give a less stable duplex than without the pairing.
|B||C, G, T||V|
|D||A, G, T||H|
|H||A, C, T||D|
|V||A, C, G||B|
|N||A, C, G, T||N|