The importance of melting temperature in molecular biology applications

The melting temperature of an oligonucleotide duplex, or T_m, is the temperature at which half of the oligonucleotide molecules are single-stranded and half are double-stranded, i.e., the oligonucleotide is 50% annealed to its exact complement. T_m is a critical parameter to consider when designing and performing many molecular biology experiments, including PCR and qPCR.

Selecting primer melting temperature

Accurate prediction of T_m identifies duplexes that are likely to form at specific temperatures, allowing you to determine appropriate thermal cycling parameters. During the annealing phase of PCR, the reaction temperature needs to be sufficiently low to allow both forward and reverse primers to bind to the template, but not so low as to enable the formation of undesired, non-specific duplexes or intramolecular hairpins, both of which reduce reaction efficiency. Both primers in PCR should be chosen to have a similar T_m. IDT recommends selecting an annealing temperature 5–7°C below the lowest primer T_m.

Selecting probe melting temperature

Designing qPCR assays with dual-labeled probes also requires careful coordination of primer T_m. When the reaction temperature is lowered from denaturing to annealing during cycling, the probe needs to anneal first to the target. If the probe binds to the target at the same time or after the primers bind, the polymerase may begin replication of target that does not contain bound probe. As a result, new DNA will be synthesized without associated probe degradation and, therefore, will not be detected as an increase in fluorescence. Such a situation leads to inaccurate data. For standard qPCR, IDT recommends a probe that has a T_m 5–10°C higher than the T_m of the primers.

Double-check published T_m data

It is important to check the T_m of any oligonucleotide sequences used in PCR even when a previously successful primer and probe set is taken directly from a publication. The design of such published sequences may incorporate T_m enhancers such as a minor grove binder or locked nucleic acid bases. These T_m enhancers are not necessary for gene expression analysis; unmodified probe and primer sets that provide reliable, accurate data can be designed for the same targets without the added expense of unnecessary modifications.

Predicting T_m  (Melting Temperature)    

What is an efficient and reliable way to predict T_m? The OligoAnalyzer tool from IDT—found in the SciTools applications section of our website―as it is the result of continuing research and innovation. It takes into account the effects of oligonucleotide, cation, dNTP, and salt concentrations, oligonucleotide sequence, and nearest-neighbor interactions. The consideration of all these factors enables accurate prediction of oligonucleotide T_m specific to your reaction conditions [1].

For more information on the calculations and algorithm used by the OligoAnalyzer tool, refer to the IDT technical report, Calculation of T_m for Oligonucleotide Duplexes.