Well, that's going to be complicated. Not a lot of divergence in mtDNA sequences between closely-related species.

As far as "how many SNPs before a primer/probe won't bind", the answer is that you have to figure it out empirically. Mismatches at the 5' end do not matter. Mismatches at the terminal 3' base can (but do not always), block extension. Mismatches in the middle will give a reduced efficiency, but not likely to be 0 amplification.

qPCR products need to be very small (80-120 bases) and hard enough to get a set with a high efficiency without adding in yet another parameter.

Honestly, unless you have 100s of samples, it's going to be faster and easier to just use Sanger sequencing. The amount of time you'll spend trying to get the qPCR assay optimized is a LOT. qPCR reagents are costly, Sanger is cheap.

Good luck!

Dear Stephanie S Coster ,

I do not agree with Katie, since SNP characterization can be done in the human genome with TaqMan Probes differentiating single SNPs it should be possible to set up a custom TaqMan primer/probe combination to differentiate the two species. I have used a jak2 v617f pair to differentiate and was even able to create a standard (with mixed cloned PCR fragments) to differentiate between the a mounts of cells carring the SNP.

You should try a design tool like this:

https://www.thermofisher.com/order/custom-genomic-products/tools/genotyping/

Best wishes Soenke

1–2 SNPs can block primer/probe binding, especially at the 3' end

There are several factors to consider to ensure the accuracy of the reaction:

There must be 3 single-nucleotide mutations in each primer region: Because mutations are very few or in regions that may not significantly affect primer binding, they may not significantly affect primer binding.

Using the same primer with 3 different mutations in the probe may work. Using the same primer with a probe distinguished by mutations may work if the mutations affect the probe's binding efficiency.

The number of single mutations required depends on the ability of the primer and probe to successfully bind to the target sequence. Having more than 3 mutations in a small region can result in weak or no binding to the target protein or DNA, making the reaction less accurate.