The melting temperature of an oligonucleotide duplex, or Tm, 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. Tm 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 Tm 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 Tm. IDT recommends selecting an annealing temperature 5–7°C below the lowest primer Tm.
Selecting probe melting temperature
Designing qPCR assays with dual-labeled probes also requires careful coordination of primer Tm. 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 Tm 5–10°C higher than the Tm of the primers.
Double-check published Tm data
It is important to check the Tm 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 Tm enhancers such as a minor grove binder or LNA bases. These Tm 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.
What is the best and easiest way to predict Tm? The OligoAnalyzer® tool from IDT, found in the SciTools applications section of our website, 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 Tm specific to your reaction conditions . For more information on the calculations and algorithm used by the OligoAnalyzer tool, refer to the IDT technical report, Calculation of Tm for Oligonucleotide Duplexes.