Primer Inspector

Introduction

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.

Primer Sequence Input

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.

Settings

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:

  • Primer Concentration: The dissociation equilibrium and hence the melting temperature depends on concentrations of the annealing oligo- and polynucleotides.
  • Total monovalent cation concentration: 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).
  • Mg2+concentration: Magnesium cations strongly affect DNA duplex stability so their concentration must be provided in order to estimate the melting temperature accurately.
  • Total dNTPs concentration: 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.
  • Tm 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.

To trigger the analysis, click the Apply & Analyze button. To erase all fields, click the Clear All button.

Understanding the Results

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 [2] and [3]). All the melting temperature calculations used in the app are based on the nearest neighbor method with parameters described in references [1] and [4].

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 [4]. 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.

References

1.

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.

Pubmed

2.

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.

Pubmed

3.

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.

Biochemistry. 2008;47(19):5336-53.

Pubmed

4.

The thermodynamics of DNA structural motifs

SantaLucia J. Jr., Hicks D.

Annu Rev Biophys Biomol Struct. 2004;33:415-40.

Pubmed