Table 2 Summary of different approaches utilized for each genetic situations.

No common SNP in the amplicon

Direct HRM analysis

Only one common SNP in the amplicon

Sometimes it is possible to distinguish directly the three groups (Figure 1)

Otherwise, spike-in or unlabelled probe is needed (Figure 2)

Two SNPs in the amplicon, one is common the other is rare

Spike-in when possible (Figure 3); otherwise, use unlabeled probe for the common SNP, and perform mutation scanning for the rare one (Figure 4)

Two or more common SNPs in the amplicon

Use an unlabeled probe for each SNP (Figure 5)

Two SNPs in two different amplicons

When T_m of 2 amplicons is different, it is possible to perform multiplex PCR (Figure 6)