Are the Tm and Ta values of PCR primers based on entire sequences or just annealing portions?

Question

I am trying to use the Q5 mutagenesis kit to produce an indel mutation in my protein.

I used the NEB BaseChanger interface to design my primers (https://nebasechanger.neb.com/). However, I am confused about the way NEB BaseChanger calculates the Tm and Ta of these primers. When it generates the primers, it indicates the Tm and Ta are in the low 60s. However, when I put the same primers into the NEB Tm calculator, I found that their Tm is in the high 80s.

I suspect this is because the NEB BaseChanger is not counting non-annealing bases (bases that encode the inserted amino acids). I am wondering which website to trust - I understand that if the Tm and Ta are what the NEB Tm calculator suggests they are, my PCR will likely not work. But if the Tm and Ta are what the BaseChanger suggests, the reaction should run.

Thanks for any and all advice!

Answer 1

The insertions don't count towards the annealing temperature, at least for the first and most critical cycle of the PCR. After the first cycle, where the bases get incorporated into the product, for the second and subsequent cycles these bases should matter, but the primers should match the target 100% (because of the incorporated primer) and a lack of efficiency caused by the high Tm doesn't matter because of the binding specificity.

Remove the non-annealing insertion bases before calculating the annealing temp.