The best location to place the mutation for discrimination is very near the 3' end of one of the primers. The last 3 bases are particularly important for extension from a primer. If you are not using a proof-reading polymerase, I would generally use the second to the last base, but it may depend on the specific primer sequence context.

If you are using something like Taqman, you might be able to put the mutation under the probe, or even a pair of different colored probes designed to discriminate the mutation. I haven't done this, but would probably try the two most 5' bases of the probe for this.

Bio-Rad have a design tool on their website. Using the link below select "Get assays designed specifically for Droplet Digital PCR" and then log in.

https://www.bio-rad.com/en-uk/product/primepcr-pcr-primers-assays-arrays?ID=M0HROA15

Their regional ddPCR specialist will be able to help too.

Thanks, but I think I cannot discriminate based on the primers. The intention is to have a FAM labeled probe for mutant and a TET probe for wild type. Then quantify the relative amounts of each. I will look at the Bio-Rad website once my colleague has supplied me with the relevant sequence as it isn’t on COSMIC. I just like to be able to do these things myself without relying on Web tools!

It has been a while ago that this was posted, but for future reference it is best to position your SNP in the middle of your sequence and otherwise more in the 5' direction. However, from experience I know that it is not easy in case your wild-type and mutant only differ one mutation. Other tips are:

- Use LNA probes as they increase the Tm and consequently you can make shorter probes that are more specific

- Use probes in competition > One probe that targets the wild-type another the mutant. Due to this competition it will be more specific.

- Shorter amplicon sizes are better! Especially for ddPCR stay below 150 bp and preferably even shorter

- You could consider lowering the probe concentration in case of false positive results