This protocol provides a high throughput and reliable makeup for NHEJ analysis that can determine the prevalence and relative quantity of Indel mutations in gene-edited mosquito populations. Compared to the next generation, or Sanger sequencing, ddPCR has faster turnaround time for results, which allows quick and complete analysis of genetic variation to field for genetically modified organisms. This method should be generally applicable to all experimental systems.
Demonstrating the procedure will be Thai Pham, a staff research associate from the Yang's lab. 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 pipetteing. 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 drop 40 microliters of liquid sample for three to five seconds at an angle of 30 to 45 degrees. Then expel the mixture into the well slowly for over three 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 five 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 from one minute to anneal, and 60 degrees celsius for two minutes to extend. After 40 cycles, set hold at 98 degrees celsius for 10 minutes, and final hold at four degrees celsius.
Use a ramp rate of two 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.
Designate the correct experimental parameters for software analysis, namely sample information, super mix, target name, target type, signal channel one and channel two. Then, set the threshold for droplet count above 10, 000 for reliable results. Next, check the droplet count for each well in the droplet tab, ensuring that all are above 10, 000.
Finally, check 1D amplitude for efficient signal separation from negatives. In the plate editor, highlight the entire plate, and set the experiment type to drop off. Set the WT target as a reference, designating channel one for FAM and channel two for HEX.
Set NHEJ target as unknown, designating channel one for FAM and channel two as none. In the 2D amplitude tab, set the cluster thresholds with the graph tools for each sample. The tail is normally associated with the WT cluster for NHEJ assays.
Under the ratio tab, click on the gear icon from the top right of the graph, and select the fractional abundance to plot a point corresponding to the percentage of NHEJ events. 15 different pulled samples of 10 mosquitoes each contained various NHEJ alleles, were analyzed and identified using the drop off assay, and results showed that all 15 samples carried 100%Indel alleles. In another experiment, 11 pulled samples of wild type mosquitoes and NHEJ mosquitoes with different NHEJ percentages were examined with this ddPCR protocol.
The results showed that the identified percentage was comparatively close to the Indel detection by amplicon analysis technique. Pipette slowly to avoid bubbles, and allow all the emulsified droplets to drip down the wall of the wells.
