Digital Droplet Polymerase Chain Reaction for Indel Mutation Detection in Target Genes

1. ddPCR reactions and droplet generation preparation

1. Quantify DNA using a fluorometer.
   NOTE: For the drop-off assay, it is recommended to use a range of 3,000-30,000 haploid genome copies per reaction, which is designed to detect NHEJ events with a HEX-labeling probe that binds to a WT sequence of the targeted cut site and will not anneal (drop-off) if there is a deletion or insertion at the target site, indicating the presence of an NHEJ variant.
2. Calculate the copy number using the haploid genome weight and concentration of DNA in the extract. This is done by multiplying the concentration of the extracted DNA by the volume used to obtain the total DNA mass, then dividing it by the haploid genome weight. Ensure that the volume added is between 1-10 µL. Dilute as necessary to be in the recommended haploid genome copy range.
   NOTE: One Anopheles gambiae haploid genome is estimated to be 0.27 pg per adult mosquito.
3. Design primers and probes. Design forward and reverse oligonucleotide primers with a Primer Melting Temperature (Tm) in the range of 55-60 °C that flank the 5'- and 3'-ends of the gRNA target site producing an amplicon of 150-400 bp.
   1. HEX (Hexachloro-fluorescein)-labeled probe for NHEJ detection: Design an oligonucleotide of ~15-20 bp in length complementary to the target site and add the HEX-probe to the 5'-end and BHQ1 (BLack Hole Quencher 1) to the 3' end. The Tm of the hydrolysis probe should be 3-10 °C higher than the Tm of the primers.
   2. FAM (6-carboxyfluorescein)-labeled probe for reference WT: Design an oligonucleotide ~15-20 bp in length complementary to a conserved genome site distant (about 25 bp) from the target site and add the FAM-probe to the 5'-end and BHQ1 to the 3' end. The Tm of the hydrolysis probe should be 3-10 °C higher than the Tm of the primers.

2. PCR reaction preparation

1. Prepare 25 µL of the ddPCR Sample Mix with the following components: ddPCR supermix for probes (no UTP): 12.5 µL, forward and reverse primers (10 µM): 1 µL each, HEX/FAM probes (10 µM): 0.625 µL each, DNA: 1-5 µL (3,750-37,500 haploid genome copies) and water: up to 25 µL.
2. Thoroughly mix the reactions by vortexing or reflux pipetting (up and down) (20x).
   NOTE: If the reactions are in a 96-well plate, pipette the entire volume up and down 20 times rather than vortexing to avoid bubble formation.
3. Briefly centrifuge the samples to settle the mixture at the bottom of the tube or well.
   NOTE: Ensure the reactions are at room temperature for the droplet generation. Prepare 1x of ddPCR mix for extra/unused wells in each cartridge (each cartridge has 8 wells).

3. Droplet generation

1. Using a 50 µL multichannel pipette, load 20 µL of the ddPCR sample mix into the middle row of the cartridge (Figure 1A, top).
2. Load 70 µL of the oil into the bottom row. Load 20 µL of 1x ddPCR supermix into unused wells.
   NOTE: Do not introduce bubbles.
3. Place the gasket touching only the edges, avoiding the center concaved area (Figure 1B).
4. Place the plate securely in the droplet generator and close the cover to start the run.
5. Using the multichannel pipette, transfer 40 µL of the emersion mix from the top row of the cartridge (Figure 1A, bottom) into the 96-well plate.
   1. Draw the liquid sample for 3-5 s at a 45-30° angle. Expel the mixture slowly for over 3 s at a 45° angle into the side of the well, allowing it to drip down the side. It is okay to go to the second stop (complete expulsion) of the pipette to expel all the liquid.
6. Using foil heat seals, seal the plates for 5 s at 180 °C.

4. PCR

1. Place the sealed plate into the thermocycler (Figure 1C) and set the recommended PCR conditions if following the NHEJ Drop-Off guidelines as follows:
   1. Initial denaturation at 95 °C for 10 min.
   2. Set 40 cycles of 94 °C for 30 s to denature, 55 °C for 1 min to anneal, and 60 °C for 2 min to extend.
   3. Hold at 98 °C for 10 min.
   4. Hold at 4 °C.
      NOTE: The annealing temperature for specific primers and probe sets may be optimized using a thermal gradient. Use a ramp rate of 2 °C/s for all the steps. PCR conditions should be adjusted depending on each experimental design and set-up.

5. Droplet reading

1. Place the plate securely in the droplet reader with well A-1 at the top left (smoothed corner, the other three are edged) (Figure 1D).
2. Set up the plate in the program: Designating FAM as the known reference channel and HEX as the unknown one (Figure 2A).
3. Run the Droplet reading experiment as direct quantification. After the run finishes, change the Experiment type to Drop-Off (DOF) for the analysis (Figure 2A).