At Tm, the equilibrium between bound and unbound primers is such that about 50% of primers are bound. By setting the temperature lower, this becomes closer to 100%. Further lowering of temperature will result in unspecific binding to sequences that differ in one or more nucleotides.

At Tm, the equilibrium between bound and unbound primers is such that about 50% of primers are bound. By setting the temperature lower, this becomes closer to 100%. Further lowering of temperature will result in unspecific binding to sequences that differ in one or more nucleotides.

I agree with Matej. Lower temperature will result non-specific bind.High temperature will not allow primers to bind to target DNA.

hello Adam

i will try to put it in simple term for you to understand.

When we say denaturation (melting) it means making a double strant DNA to single strand at 95 C. But primers are oligonucleotide (approx 20 bp), the melt at temperatures before 95 C. each primer will have different tm (melting) temp based on the GC%(nucleotide content, based on double or triple bonds).

In your question u asked why we need to have annealing temp 5C lower than melting temp(tm). Annealing a process where primer binds to the template and melting is atemperature when they denature. so reducing the 5C from the melting will allow it to bind. Why only 5C why not 10C or lower? the problem is it will lead to unspecific binding. since we donot want unspecific amplification we stick to 5C lowering temp. but if u design a primer which you say it is so specific  even at lower temp it wont bind to any other place then u can use lower annealing (highly unlikely).

I guess it answered your question.

Good day.

If you have a gradient thermal cycler, usually it's better to just to do a range of Ta plus/minus 10°C of the calculated Tm and see the best temperature.

Actually, quite often there is not much difference.

There are also plenty of ways to calculate Tm itself. I expect the online IDT OligoAnalyzer to be the most reliable since they have performed many experimental calibrations.

Depends on what you're up to, Adam: For amplifying something off a (pure) plasmid or from a PCR product, annealing temp is pretty much uncritical. When you need to amplify a transcript from a mRNA prep or when you have the whole genome in your pot, then you might need to do a lot of optimization work (annealing temp, annealing time, touch down PCR, just to name some parameters to tweak). The calculated Tm is just a starting point for this and also a guideline for designing primer pairs with similar properties.