Standardization of annealing temperature for Single-variant specific PCRs
A gradient PCR (SimpliAmp Thermal Cycler, Applied Biosystems, USA) was performed for each of the variant separately with freshly prepared cDNA template to standardize the annealing temperature. Couple of distinct tubes was prepared for each of the variant using the respective primer pairs to differentiate between the wild type and the mutant. The PCR was carried out in 10µl reaction volume containing 3-5 ng/µl DNA, 5µl master mixture (GoTaq® G2 Green Master; Promega, USA), 0.2µM of each forward and reverse primer and 2.8 µl nuclease free water. The thermocycling conditions were as follows: initial denaturation at 95°C for 1 min followed by 30 cycles at 95°C for 30s, annealing at a range of 55-65°C for 30s and 72°C for 30s followed by a final extension at 72°C for 5min. The PCR products were electrophoresed on a 1% (w/v) agarose gel stained with ethidium bromide (UltraPure™ Ethidium Bromide, 10 mg/mL; Thermo Fisher, USA) and visualized using a gel documentation system (Bio-Rad, USA).