High resolution melt software

Genomic DNA and DNA from FA-SAT clones, both of high purity, were analysed by High-Resolution Melting Assays (HRM) with MeltDoctor HRM Master Mix, which uses the SYTO9 dye (Applied Biosystems, Thermo Fisher Scientific), and with the 83-bp B and 83-bp C primers (supplementary table S1, Supplementary Material online). All the PCR mixtures were done according to the recommendations of the manufacturer. Experiments were performed in the StepOne real-time PCR system (Applied Biosystems, Thermo Fisher Scientific) with the next program: initial denaturation at 95 °C (10 min), 40 cycles of 95 °C for 15 s, followed by 59 °C for 1 min. Subsequently, a melt curve was performed with a denaturation step of 95 °C for 15 s, an annealing of 59 °C for 1 min and a high-resolution melting of 95 °C for 15 s with a continuous ramp rate of 0.3%, followed by a final step of annealing of 60 °C for 15 s. All reactions were performed in triplicate, and negative controls (without template) were also included on the plate. The StepOne software (version 2.2.2, Applied Biosystems, Thermo Fisher Scientific) was used for data acquisition and the High Resolution Melt software (version 3.0.1, Applied Biosystems, Thermo Fisher Scientific) for data analysis (we used the standard software parameters suggested by the manufacturer).