1. HRM Assay and Optimization 1. DNA Quality 2. Amplicon LengthAmplicon  lengths of 100–300 bp 3. Primer Selection 4. Dye Selection 5. MgCl2 Concentration  MgCl2 concentration—start between 1.0–3.0 mM 6. HRM Analysis Software  Primer Tm should be between 58–60°C  Primer concentration—start at 300 nM  Provide a view of the raw fluorescent melt data  Provide a process to align that data  View melt curve differences between samples

2. Steps of HRM:  The first step of the HRM methode is the amplification of the region of interest, using standard PCR techniques, in the presence of a specialized double-stranded DNA(dsDNA) binding dye. This specialized dye is highly fluorescent when bound to dsDNA and poorly fluorescent in the unbound state. This change allows the user to monitor the DNA amplification during PCR.  After completion of the PCR step, the amplified target is gradually denatured by increasing the temperature(50°C up to around 95°C). in small increments, in order to produce a characteristic melting profile; this is termed melting analysis. The amplified target denatures gradually, releasing the dye, which results in a drop in fluorescence. When set up correctly, HRM is sensitive enough to allow the detection of a single base change between otherwise identical nucleotide sequences..  The Tm is defined as the temperature at which 50% of the DNA sample is double stranded and 50% is single-stranded. The highest rate of fluorescence decrease is generally at the melting temperature of the DNA sample (Tm). The Tm is typically higher for DNA fragments that are longer and/or have a high GC content.

3. HRM REAL TIME PCR PCR cycling and HRM analysis was performed on the Rotor-Gene™ 6000 (Corbett Research) and 5x HOT FIREPOL® Eva Green® HRM Mix kit. The HRM real time PCR master mix was prepared in final volume of 10 L for n+1 samples (including negative control).

4. After preparing master mix, the 0.1 micro tubes specialized for Corbett were placed in 72 well's thermo cycler and real time PCR programed The HRM real time PCR program is consisted of an initial activation step at 95 ºC for 15 min; followed by a 40-cycle program.