1. Droplet Digital PCR with EvaGreen Assay: Confirmational Analysis of Structural Variants
- Author
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Michael Parfenov, Joshua M. Gorham, and Angela C. Tai
- Subjects
0301 basic medicine ,Oligonucleotide ,Chemistry ,Microfluidics ,Structural variant ,Computational biology ,DNA ,Polymerase Chain Reaction ,Polymorphism, Single Nucleotide ,Article ,03 medical and health sciences ,030104 developmental biology ,Molecular Probes ,Genetics ,Nucleic acid ,Humans ,Digital polymerase chain reaction ,Lower cost ,Biological Assay ,Genetics (clinical) - Abstract
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.
- Published
- 2018