HRM versus Classical Melt Curve Analysis

HRM technology outperforms classical melt curve analysis
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In contrast to classical melt curve analysis, HRM provides significantly more information, down to single nucleotide differences. The main purpose of a melt curve is to verify the specificity of amplification and to check for the presence of nonspecific amplification products, such as primer dimers. In HRM analysis, the melting curve analysis serves to distinguish between several PCR amplicons with subtle changes in sequence and length, down to the single nucleotide level (see figure Differences between classical melt curve analysis and HRM analysis).

Differences between classical melt curve analysis and HRM analysis
Classical melt curve HRM analysis
Monitors melting differences >1 °C Monitors melting differences <0.5°C
Moderate amount of data points per °C High density of data points per °C
Main application: finding nonspecific amplicons (e.g., primer dimers) Main application: identification of DNA differences in genotyping, methylation analysis, etc
Can be done with any real-time cycler Requires special instrument and software (e.g., Rotor-Gene Q)
 Part of the experiment is performed using real-time PCR chemistry with a nonsaturating dye (e.g., SYBR Green) Needs optimized chemistry with saturating dyes (LCGreen, EvaGreen)