PCR Primer Inspector is an online application made to calculate a predicted melting temperature of a PCR primer and to analyze its propensity to form homo- or heterodimers under PCR reaction conditions.
One or more primer sequences are supposed to be provided by the user in the "Primer Sequences" field. The app accepts both uppercase and lowercase DNA sequences and is tolerant to spaces. It also accepts "N"s but not other ambiguity symbols. A presence of any character other than A, C, G, T or N will issue an error. The user can also provide primer names in the "Names" field, one per line, in parallel order with respect to the primer sequences. Missing names will be substituted automatically with Oligo_1, Oligo_2, etc.
The stability of a DNA duplex is determined by a number of factors including pH, concentrations of the duplex individual components, salts and other solutes, and of course temperature. To properly estimate the melting temperature of a PCR primer, following parameters are required:
To trigger the analysis, click the Apply & Analyze button. To erase all fields, click the Clear All button.
The output of the analysis consists of a table showing the predicted melting temperatures of the input primers and a listing of potential primer dimers. There are two melting temperatures displayed for each primer comparing results of two different algorithms for monovalent and magnesium cation correction (references  and ). All the melting temperature calculations used in the app are based on the nearest neighbor method with parameters described in references  and .
Primer dimers are ordered by predicted stability scores and pairing alignments of the oligo duplexes are depicted. 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.
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.