Droplet Digital PCR with EvaGreen Assay: Confirmational Analysis of Structural Variants

We describe a variation of droplet digital PCR (ddPCR) for DNA structural variant analysis, where expensive fluorescent probes are replaced by low-cost EvaGreen, a DNA binding dye, hence reducing cost without reducing sensitivity. ddPCR is a method that employs water-oil microfluidics and fluorescence technology to quantify target nucleic acids with extreme precision and sensitivity. Traditional ddPCR uses standard Taqman probe assays to determine target DNA concentrations, however these two-color fluorescent oligonucleotide probes are expensive and require extensive optimization. EvaGreen’s DNA binding properties allow target products to be effectively quantified at a significantly lower cost. This protocol employs the EvaGreen ddPCR assay to confirm structural variants on a ddPCR system and produces results comparable to the standard, far more expensive Taqman approach.