Droplet Digital Polymerase Chain Reaction: A Method to Generate Nanodroplet PCR Reactions for Detecting Rare Tumor Mutations

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.

Before beginning the droplet digital PCR procedure, clean the bench space and equipment with 10% bleach and 70% ethanol and gently vortex and briefly centrifuge all of the reagents except the droplet-stabilizing oil. Confirm that the compressed nitrogen gas cylinder is attached to the droplet-generating instrument and that the cylinder tank is set at 90 pounds per square inch. Add 38 microliters of digital PCR reaction mixture directly into the bottom of each well of an 8-well PCR tube strip removing any bubbles with a clean pipette tip.

Add 12 microliters of pre-amplified DNA sample in triplicate to the appropriate wells of the tube strip. Gently pipette the full volume 10 times to mix the reaction mixture with the DNA sample and pipette the full volume from each well into the corresponding A through H channels of a droplet-generating instrument chip. Insert a new PCR tube strip into the droplet-generating instrument and scan the droplet-generating instrument chip ID into the software on the instrument computer.

Then, click "Start the run" to begin dropletizing samples. When the dropletization is complete, remove the PCR tube strip and apply tube strip caps. Transfer the tube strip to a thermal cycler and balance the tube with another tube strip containing 80 microliters of water per well. Then, run the thermal cycler under the appropriate thermal cycling conditions. Replace the strip caps with high-speed caps.

When the thermal cycling is complete, transfer the tube strip to the quantification instrument. Click "Setup a run" on the instrument software and place the tube strip into the quantification instrument. Place the metal shield on top of a new quantification instrument chip. Scan the chip ID into the instrument and insert the chip into the machine. Close the lid of the instrument. In the computer software, enter a name for the digital PCR run and for each of the eight channels. Then, select "Fast mode" and click "Start" to begin the quantification.