deus ex computa
     Skip Navigation Links.

Base pair mismatch

Biomaths an Android application which contains this function can be found here on the Android Market Place / Google Play.

Purpose of this page

This web page is designed to give you an idea of the effect of single base pair mismatches and not used to calculate a primers Tm. There are many programs that claim to do this, but compared to RNA secondary structure very little is known about the thermodynamics of DNA base pairing. When in the lab I design primers such that they have a length of 20bp and more then 9 C:G base pairs. This is simple and works just as well as anything else I have used. However when designing ARMs primers for instance, it is important to understand that in some instances mismatched bases will hydrogen bond with a greater strength than a A:T base pair. Also the notion that two mismatched purines will be less favourable than two smaller pyrimidines is just plain wrong. In some instances G:G base pairing is energetically favourable where as some C:C base pairs are not.

The web page will not work correctly for consecutive sequence mismatches since knowing if the sequence forms a bulge, bubble or mismatch is almost impossible to say and any resultant structures are difficult to model.

Oligonucleotide sequence (maximum length 50bp)


Target sequence (maximum length 50bp)






Enter the sequence of the oligonucleotide in the upper text box (i.e. acgctagcggctagctag) and the target sequence (i.e. acgctagcggctacctag) in the second text box. The program expects the two sequences to be the same length. Once both sequence have been entered press the submit button.

About the calculation

The system uses the free energy values for pairs of base pairs, i.e. 5'-CpA-3' hydrogen bonding to 3'-GpT-5'. When this is done experimentally, Tm results match calculated Tm values better than using single base pairs, while using three base pairs does not increase the accuracy.

Most of the experimental work to calculate the free energy of the base pair mismatches was done using relatively small oligos at high salt concentration compared those used in PCR or sequencing. Consequently like all other Tm calculation methods it's best to use the values with care.

If you want to learn more about mismatch base pairing try:

Santa Lucia J Jr, Hicks D. (2004)
The thermodynamics of DNA structural motifs.
Annu Rev Biophys Biomol Struct. 33:415-40.

Here is the PubMed link.

Copyright © 2020 Insilicase.