This article discusses the use of digital droplet PCR (ddPCR) for detecting indel mutations in DNA. The method partitions a sample into tiny droplets for independent amplification, allowing for precise mutation analysis.
Digital droplet PCR detects indel mutations — random insertion or deletion of nucleotides in the DNA. This is achieved by partitioning the sample into tiny droplets and independently amplifying the DNA in each droplet.
To perform ddPCR, take a DNA sample containing wild-type and mutant variations of the target gene. Load the sample mix and oil into distinct compartments of the droplet generator chamber containing the microfluidic channel.
The sample and oil flow rapidly through the narrow channels, emulsify at the junction, and are partitioned into several droplets, each containing a few DNA molecules.
Now, take a PCR plate with the buffer containing forward and reverse primers, dNTPs, thermostable DNA polymerase, and two probes tagged with different fluorophores and quencher molecules. Each probe is specific to a particular type of allele — wild-type or mutant one.
Transfer the droplets to the PCR plate. Place the plate in a thermocycler, and run the reaction. During denaturation, the DNA strands separate. At annealing temperature, the primers and probes anneal to the template DNA.
The probe with green and orange fluorophores binds to the wild-type and mutant DNA sequence respectively. Later, DNA polymerase extends the primers, cleaving the fluorophore and quencher molecules from the probe, generating a fluorescence signal.
Analyze the amplified product. The droplets showing green fluorescence contain the wild-type DNA sequence, while the orange fluorescence indicates the indel mutation.
To begin with, prepare 25 microliters of the digital droplet PCR or ddPCR sample mix by adding ddPCR super mix, forward and reverse primers, HEX or FAM probes, DNA, and water in the proportions described in the text. Then, thoroughly mix the reaction by vortexing.
Using a 50-microliter multichannel pipette, load 20 microliters of the ddPCR sample mix into the middle row of the cartridge. Then, use a 200-microliter multichannel pipette to load 70 microliters of the oil into the bottom row without generating bubbles throughout the pipetting.
Place the gasket, touching only the edges, avoiding the center concaved area, and then, place the plate securely in the droplet generator, and close the cover to start the run.
To transfer the immersion mix from the top row of the cartridge into the 96-well plate, use a multichannel pipette and draw 40 microliters of liquid sample for 3 to 5 seconds at an angle of 30 to 45 degrees. Then, expel the mixture into the well slowly for over 3 seconds at a 45-degree angle, allowing it to drop down from the side, and then, go to the second stop of the pipette to expel the liquid completely. Using foil heat seals, seal the plates for 5 seconds at 180 degrees Celsius.
Place the sealed plate into the thermocycler and set the PCR conditions for NHEJ Drop-Off guidelines by setting initial denaturation at 95 degrees Celsius for 10 minutes. Then, set 40 cycles of 94 degrees Celsius for 30 seconds to denature, 55 degrees Celsius for 1 minute to anneal, and 60 degrees Celsius for 2 minutes to extend.
After 40 cycles, set hold at 98 degrees Celsius for 10 minutes, and final hold at 4 degrees Celsius. Use a ramp rate of 2 degrees Celsius per second for all steps. The annealing temperature will vary according to the probes or primer used. Place the plate securely in the droplet reader with the A1 labeled well at the top left.
Open the program and set up the plate by designating FAM as the known reference channel and HEX as the unknown one for each well. Then, change the name for each sample. Run the droplet reading experiment as direct quantification while saving the template.
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