Spreadsheet Software for Thermodynamic Melting Point Prediction of Oligonucleotide Hybridization with and without Mismatches

Abstract

The use of thermodynamic parameters for the calculation of oligonucleotide duplex stability provides the best estimates of oligonucleotide melting temperatures (Tm). Such estimates can be used for evidence-based design of molecular biological experiments in which oligonucleotide melting behavior is a critical issue, such as temperature or denaturing gradient gel electrophoreses, Southern blotting or hybridization probe assays on the LightCycler™. We have developed a user friendly program for Tm calculation of matched and mismatched probes using the spreadsheet software Microsoft Excel®. The most recently published values for entropy and enthalpy of Watson-Crick pairs are used, and salt and oligonucleotide concentrations are considered. The 5′ and 3′ end stability is calculated for the estimation of primer specificity. In addition, the influence of all possible mutations under a given probe can be calculated automatically. The experimental evaluation of predicted Tm with the LightCycler, based on 14 hybridization probes for different gene loci, showed an excellent fit between measured results and values predicted with the thermodynamic model in 14 matched, 25 single mismatched and 8 two-point mismatched assays (r=0.98; Sy.x=0.90; y=1.01×–0.38). This program is extremely useful for the design of oligonucleotide probes because the use of probes that do not discriminate with a reasonable Tm difference between wild-type and mutation can be avoided in advance.