High Resolution Melting (HRM)

High Resolution Melting (HRM) analysis is a relatively new, post-PCR analysis method used to identify variations in nucleic acid sequences. The method is based on detecting small differences in PCR melting (dissociation) curves. It is enabled by improved dsDNA-binding dyes used in conjunction with real-time PCR instrumentation that has precise temperature ramp control and advanced data capture capabilities. Data are analyzed and manipulated using software designed specifically for HRM analysis.

High Resolution Melting (HRM) analysis is an alternative to dHPLC sequencing screening of new gene variants. The HRM Software is now available on the 7500 Fast, StepOnePlus, and StepOne Real-Time PCR Systems. The StepOne (48-well) and StepOnePlus (96-well) Real-Time PCR Systems are remarkably simple systems designed with a user-friendly, yet powerful, interface for researches of all experience levels. The 7500 Fast Real-Time PCR System delivers precise results with fast thermal cycling in a standard 96-well format. The AB HRM application does not require temperature shifting, which results in a greater likelihood of identifying new homozygous mutations than methods that require temperature shifting.

Figure 1: Difference plot generate on another plate based real-time HRM system.

The ability to easily identify new variants is key for successful HRM applications. An HRM system from Competitor R (Fig 1) was only able to distinguish them in 10% of the population.

By eliminating the temperature shift step, the Applied Biosystems HRM solution (Fig 2) was able to clearly distinguish homozygous variant samples from homozygous wild type samples in 97.5% of the population.

All genotypes were auto-called by the respective software packages and were not altered by the operator. Class 1 SNP (A/G), multiple technical replicates of nine DNA samples representing three genotypes: homozygous wildtype (G/G), homozygous mutant (A/A) and heterozygous (A/G).