The CADMA cycling protocol was initiated by one cycle at 95°C for 15 min, followed by 45 cycles of 95°C for 10 s, annealing temperature (TA) for 20 s (Table 1), 72°C for 20 s, and one cycle at 95°C for 1 min. HRM was performed from 65°C to 95°C with a temperature increase of 0.1°C/s.
The cycling parameters were 40 cycles of 94°C for 1 min and 50 60°C (depending on the primers) for 1 min, followed by one cycle at 72°C for 10 min. The amplified products were electrophoresed using the HEGS system.
The PCR was completed by one cycle at 72°C for 5 min.
The AmpliTaq gold was activated by one cycle at 95 °C for 10 min and cDNA amplified for 35 cycles of 95 °C for 30 s, 60 °C for 30 s and extension at 72 °C for 1 min.
Thus, the 45 cycles for cDNA amplification were followed by one cycle at 95° for 1 min, one cycle at 55° for 1 min, and 80 cycles at 55° for 10 sec.
Finally, a dissociation curve to test PCR specificity was generated by one cycle at 95°C (15 s) followed by 60°C (20s) and ramp up to 95°C with acquired fluorescence during the ramp to 0.01°C/s.
The reaction mixture was subjected to 35 amplification cycles, each consisting of denaturation at 94°C (60 s), followed by one cycle, annealing at 94°C (20 s), 67°C (2 s) followed by seven cycles and extension at 93°C (5 s), 65°C (4 s), with a final extension in step with 28 cycles.
Polymerase chain reaction parameters were one cycle at 95 °C for 5 min, then 35 cycles of 94 °C for 1 min, 48 °C for 45 s, 72 °C for 2 min followed by one cycle of extension at 72 °C for 10 min. Denaturing gradient gel electrophoresis was performed using the DCode Universal Mutation Detection System (Bio-Rad Laboratories, HerCAles, CA, USA).
The PCR amplification were done using 1 cycle of 3 min at 94 °C, followed by 35 cycles of 1 min at 94 °C, 2 min at 53 °C and 2 min at 72 °C, followed by one cycle of 7 min at 72 °C in a thermal cycler (Model 2720, Applied Biosystems, USA).
Thermocycler conditions were: 15 min at 95° to activate the enzyme and denature the DNA, 40 cycles of 1 minute denaturation at 94°, 1 minute annealing at 55°, 1.5 minute extension at 72°, followed by one cycle of 10 minute extension at 72°.
Amplification was performed using a PTC 150 thermal cycler (MJ research, Waltham, MA, USA) with the following settings: one cycle of 10 min at 94°C, followed by 30 cycles of denaturation at 94°C for 30 sec, annealing at 55°C for 2 min, extension at 72°C for 2 min, and by one cycle of final extension at 72°C for 5 min.
