Gradient Polymerase Chain Reaction to Determine the Optimum Annealing Temperature

1. Scanning for single nucleotide polymorphisms (SNPs), HRMA primer design, and primer validation

1. SNP identification in wild-type laboratory colony mosquitoes
   1. Select the target exon to disrupt proper polypeptide translation.
      NOTE: The target should be close to the start codon or amongst key residues required for protein function. The shorter the exon (e.g., ≤200 bases), the more difficult it is to target and analyze. Avoid editing close to the boundaries of an exon, as this forces one of the HRMA primers to either cross an intron or be in an intron. This is undesirable because SNP rates tend to be much greater in those regions.
   2. Primer design
      1. Go to the NCBI Blast – Primer Blast website, copy and paste the chosen exon in the box at the top of the page | choose the PCR product size (ensure it encompasses most of the exon) | choose the organism.
      2. Click on Advanced parameters | Opt (for PCR Product Tm) and add 60 (for an optimal temperature of 60 °C) | Get Primers. Keep the other parameters as default.
   3. Obtain genomic DNA (gDNA) from the wild-type laboratory colony. Set aside 10 mosquitoes, anesthetize them with CO₂, and place them in a Petri dish on ice to keep them inactive. Set up 10 tubes containing 0.5 µL of the reagent for the release of DNA from tissue and 20 µL of dilution buffer, both provided in the DNA release kit suggested in the Table of Materials.
   4. Remove one leg from a single mosquito using forceps and place it in a corresponding tube of a diluted solution of the DNA-release reagent (from step 1.1.3), completely submerging the leg in the solution. Repeat this step with the remaining mosquitoes, wiping the forceps with 70% ethanol before proceeding to the next one.
   5. Incubate the leg-containing solution at room temperature for 2-5 min, then at 98 °C for 2 min, and allow it to cool down while setting up the PCR reaction.
      NOTE: Plates containing the released gDNA can be stored at -20 °C, and the protocol can be paused at this point.
   6. Prepare 10 tubes containing 10 µL of 2x PCR Master Mix, primers to a 0.5 µM final concentration, and molecular grade water to a final volume of 20 µL, and transfer 1 µL of the diluted sample to each tube. Perform PCR following these cycling parameters: 98 °C 5 min, 40 cycles of 98 °C for 5 s, 60 °C 30 s, 72 °C 20 s per kb; final extension of 72 °C for 1 min.
   7. Purify the PCR products with an enzyme to degrade the residual PCR primers and dephosphorylate excess dNTPs or any column clean-up kit. Proceed with sequencing the samples.
   8. Analyze each electropherogram for the presence of double peaks/ambiguous bases, and adjust base calls manually in each sequence using the appropriate degenerate base code.
      NOTE: This step must be performed before multiple sequence alignment as it is common for the base-calling software to select the more prominent peak as the "true" base call, giving the false impression of an absence of SNPs.
   9. Perform a multiple sequence alignment using the alignment software, SeqMan Pro, listed in the Table of Materials or other open-source alignment software, such as ClustalW or T-Coffe.
      1. Open the alignment software | click on Add Sequences | select the desired sequences and click on Add | Once all sequences are chosen, click on Done.
      2. Click on Assemble to perform the alignment. To open the alignment, click on Contig 1 and analyze the alignment and identify the SNPs (Figure 1).
         NOTE: As an alternative to steps 1.1.3-1.1.6, PCR can be performed from isolated genomic DNA obtained from bulk samples (derived from >10 individuals). Sequenced amplicons can be analyzed directly for the presence of SNPs appearing as double peaks in the electropherogram, though rare SNPs will be more difficult to detect.
   10. Design 3-5 single guide RNAs (sgRNAs), avoiding regions containing any SNP identified above, following the protocol described in.

2. HRMA primer design

1. Test the exon sequence for the possible formation of secondary structures during PCR using mFold.
   1. Go to the UNAFold Web Server | click on mFold | on the dropdown menu, click on Applications | DNA Folding Form.
   2. Enter the sequence name in the box and paste the exon.
   3. Change the folding temperature to 60 °C; on the Ionic conditions, change [Mg⁺⁺] to 1.5, and on Units switch to mM; click on Fold DNA.
   4. On Output, below Structure 1, click on pdf to open the Circular Structure Plots.
2. Go to NCBI Blast – Primer Blast for primer design.
3. Copy and paste the selected exon sequence determined by sequencing in step 1.1 (the sequence that contains the lowest number of SNPs or no SNPs) in the box at the top of the page.
4. Use the symbol < > to mark and exclude sequences that contain SNPs, the target site, and regions with possible formation of secondary structure.
5. Select the PCR product size to be between 80 and 150 bp and choose the organism.
   NOTE: Larger fragment sizes can be successfully used (~300 bp). However, longer amplicons may decrease sensitivity between sequences differing in one or just a few base pairs.
6. Click on Get Primers. Select 2–3 pairs of primers to be tested (ideally, primer sites are ≥20–50 bp away from any CRISPR target sites).

3. Primer validation

1. Perform a gradient PCR using gDNA from a single individual.
   1. Prepare a master mix and remove one sample for the non-template control (NTC) in a separate tube. Add the template to the remaining master mix and aliquot into a 96-well plate.
   2. Follow the cycling parameters: 98 °C 30 s, 34 cycles of 98 °C for 10 s, 55–65 °C 30 s, 72 °C 15 s; final extension of 72 °C for 10 min.
   3. Generate thermal melt profiles following the parameters: denaturation step 95 °C for 1 min, annealing 60 °C for 1 min, melt curve detection between 75 °C and 95 °C in 0.2 °C increments, with a hold time of 10 s at each temperature.
      NOTE: Only annealing temperatures with a single thermal melt profile should be used.