Droplet Digital Polymerase Chain Reaction: A Method to Generate Nanodroplet PCR Reactions for Detecting Rare Tumor Mutations
Transcription
Droplet digital polymerase chain reaction or dPCR technology partitions reactants of a single PCR reaction into millions of nanodroplet PCR reactions. This mass partitioning followed by amplification detects a minute amount of mutant DNA against a large background of wild-type DNA, enabling early cancer detection.
To begin, aspirate dPCR master mix containing fluorescently labeled oligonucleotides along with other PCR reagents and a droplet stabilizing oil. Add this master mix directly into the bottom of each well in a PCR strip tube to avoid air bubbles. Next, add DNA samples to the reaction mixture and pipette multiple times to mix.
Now, transfer the entire reaction mixture into a specialized chip of a droplet-generating instrument. Mount a fresh PCR strip tube onto the droplet-generating instrument. The droplet generator combines the sample and oil to form discrete nanodroplets with an encapsulated area that prevents cross-contamination between the droplets.
Once dropletization is complete, transfer the PCR strip tube to a thermal cycler to amplify the target DNA. Subsequently, load the PCR product into a droplet reader containing a fluorescence detector, which counts the fluorescent positive and negative droplets. The number of positive droplets can be used to calculate the target DNA concentration in the sample.
