This protocol is a very simple and efficient way to detect mutants generated by CRISPR/Cas9 technology from numerous individuals. The two main advantages of this technique are that it can be done in a few hours and that it is applicable to a multitude of organisms. Primer design to can be challenging for certain genes and multiple rounds of primer design and verification may be required to make HRMA efficient.
To perform a gradient PCR, start by preparing a master mix. Then remove 19 microliters of the master mix for the non-template control or NTC in a 96-well plate. Then, add the template to the remaining master mix.
Aliquot 20 microliters of the sample mix into the 96-well plate. Perform PCR following the cycling parameters and then generate the thermal melt profiles using the parameters mentioned in the manuscript. After gathering all the required material, prepare a master mix with 0.5 microliters of the DNA release reagent and 20 microliters of dilution buffer per individual to be genotyped.
Using a multichannel pipet, aliquot 20 microliters of the mix into a PCR plate and leave the PCR plate on ice. Next, place the anesthetized G1 mosquitoes in a glass Petri dish to keep them sedated and place eight wild-type mosquitoes in the second Petri dish. Wipe a pair of tweezers with 70%ethanol and use the disinfected tweezers to remove one of the mosquitoes hind legs.
Then, submerge the leg in the DNA release reagent solution. Place the mosquito in the corresponding vial and close the vial with a sponge. Once done, wipe the tweezers with 70%ethanol before proceeding with removing the leg from the next mosquito until the 96-well plate is completed.
Later, seal the plate with an optical PCR plate seal and incubate the PCR plate containing the legs at room temperature for two to five minutes, followed by incubation at 98 degrees Celsius for two minutes. Allow the plate to cool down to room temperature. For PCR, prepare a master mix containing the preferred components and then use a multichannel pipette to transfer 19 microliters of the master mix into each well of the 96-well plate.
After transferring one microliter of the DNA release solution containing previously prepared mosquito DNA to the plate, seal the plate with an optical PCR plate seal. Perform the PCR with the appropriate cycling parameters to generate thermal melt profiles following the parameters described in the manuscript, then examine the melt profiles by assigning wild-type control to the reference cluster. Mark the different clusters with corresponding colors on the 96-well template.
Next, select the individuals with curves of interest and remove the selected individuals from the tubes to back cross. Blood feed the made it females, followed by collecting G2As. In the representative analysis, the sequence analysis of the Aedes aegypti's ZIP11 mutant is shown.
The electropherogram from Aedes aegypti ZIP11 heterozygous mutants indicated the nucleotide position where the indel occurred. The indel is represented by a shift from single to double peaks as polymorphic positions show both nucleotides concomitantly. At the end of the run, the number of deleted or inserted base pairs was calculated by counting the single peaks.
Mosquitoes containing mutations in the genes Aedes aegypti ZIP11 and myo-fem were genotyped and sequenced verified using the high-resolution melt analysis of HRMA. The fluorescent signals of the samples were normalized to relative values of one to zero. Additionally, each curve was subtracted from the wild-type reference and denoted the corresponding magnification.
A failed automatic cluster assignment is shown in the representative results, where a proper distinction between the groups was not made. Further, each sample was analyzed individually and assigned to the correct groups based on the similarity to reference samples of heterozygotes, homozygotes, and transheterozygotes. After performing HRMAs, sequencing the DNA from the mutated individuals is necessary to analyze the indels and identify the other mutations in the targeted sequence.
