High resolution melting assays typically involve qPCR amplification followed by a melting curve collected using a fluorescent dye. Due to the sensitivity of high-resolution melting analysis, it is necessary to carefully consider PCR cycling conditions, template DNA quality, and melting curve parameters. For accurate and repeatable results, PCR thermal cycling conditions must be optimized to ensure that the desired DNA region is amplified with high specificity and minimal bias between sequence variants. The melting curve is typically performed across a broad range of temperatures in small (~0.3 °C) increments that are long enough (~10 seconds) for the DNA to reach equilibrium at each temperature step.
In addition to typical primer design considerations, the design of primers for high-resolution melting assays involves maximizing the thermodynamic differences between PCR products belonging to different genotypes. Smaller amplicons generally yield greater melting temperature variation than longer amplicons, but the variability cannot be predicted by eye. For this reason, it is critical to accurately predict the melting curve of PCR products when designing primers that will distinguish sequence variants. Specialty software, such as uMelt and DesignSignatures, are available to help design primers that will maximize melting curve variability specifically for high-resolution melting assays.