We have found a potential patient disease causing point mutation in an exon of a gene. We are interested in the possible differences between the expression of single nucleotide variation between wt/mut. We found from Miotke et. al. 2014 paper some good advice in designing a digital droplet PCR setup from cDNA for this. In this paper the primers were tailored so that the point mutation nucleotide was located in the very 3’ end of the primer (as was the wild type allele primer, separately). To distinguish the allele expression levels two primer tails were added to 5’ ends of the primers (39 nucleotide long AT-rich or 6 nucleotide long CG-rich). This way Miotke et al could determine the fluorescent level differences between the wt/mut alleles (long amplicon 104 bp and short 71 bp) using EvaGreen.

Our questions are:

Can ddPCR method detect fluorescent level differences from longer amplicons (300bp + 5’prime 5 bp short/40 bp long tail)? Our region of interest around the SNV in cDNA template is challenging, containg long CG-rich stretches.

Do you have experience, whether this kind of a primer design results in a VERY ACCURATE attachment of primers to wt/mutant alleles? (we will check this with primers designed outside the region and the do the Sanger sequencing)

What other important issues should we consider in the assay desig?

Thank you very much in advance!!