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

1. Detection and Quantification of Target DNA Using dPCR

1. Prepare the dPCR reaction mixture for 8 wells of the PCR strip tube plus 10% extra, by adding 220 µL of genotyping master mix, 8.8 µL each of 10 µM wildtype and mutant probes, 39.6 µL each of 10 µM forward and reverse primers, and 17.6 µL of droplet stabilizing oil.
2. Gently mix the dPCR reaction mixture by pipetting the full volume up and down 10–20 times. Do not vortex or centrifuge after droplet stabilizing oil has been added to the reaction mixture.
3. Obtain a PCR 8-well strip tube and dispense 38 µL of dPCR reaction mixture directly into the bottom of each well. Remove any bubbles with a clean pipette tip.
   NOTE: PCR strip tubes that are interlocked or packed tightly together can result in static electric charges, causing coalescence and noisy signal when analyzing dPCR data. To avoid this, aliquot strip tubes into separate biohazard bags, avoid over-packing the bags, and keep the tubes from rubbing together.
4. Add 12 µL of preamplified DNA sample to the appropriate wells of the PCR strip tube. For the negative control, add 12 µL of MB H₂O.
   NOTE: Analyze cfDNA samples in replicate wells. For CSF, analyze at least in duplicate, and for plasma/serum analyze each sample in triplicate.
5. Gently pipette the full volume up and down 10 times to mix the reaction mixture with the DNA sample.
6. Obtain a new droplet-generating instrument chip and pipette the full volume from wells 1–8 of the PCR strip tube into the corresponding channels A–H in the chip. Avoid touching the bottom of the channels with a pipette tip.
7. Obtain a new clean PCR strip tube and insert into the droplet-generating instrument. Scan or manually enter the droplet-generating instrument chip ID into the software on the instrument computer. Enter names for each of the 8 channels. Click Start the run to begin dropletizing samples.
8. When the dropletization has completed, remove the PCR strip tube and apply strip tube caps.
9. Transfer the strip tube to a thermal cycler and balance with another strip tube containing 80 µL of water per well.
10. Program the thermal cycling conditions as: 95 °C for 10 min; 45 cycles of two temperatures: 95 °C for 30 s and annealing temperature for 2 min; 98 °C for 10 min; and hold at 10 °C.
    1. Set the ramp rate to 0.5 °C/s and set the sample volume to 80 µL.
11. When thermal cycling is complete, remove the strip tube and transfer to the quantification instrument. Remove PCR strip caps and replace with high speed caps.
12. Turn on the quantification instrument and the connected computer with instrument software. Launch the instrument software and click Setup a run.
13. Place the strip tube into the quantification instrument.
14. Obtain a new quantification instrument chip and scan or manually enter the chip ID into the instrument. Insert the chip into the machine.
15. Place the metal shield on top of the chip and close the lid of the instrument.
16. In the computer software, enter a name for the dPCR run and for each of the 8 channels (A–H). Select Fast mode (which must be used with high speed caps) and click Start to begin the quantification.
17. When quantification is complete, remove the metal shield (to be saved and reused) and dispose of the PCR strip tube and chip. On the instrument computer, the Run Log will contain result files for each run. Use the .fcs files for each sample to analyze with the analyst software.