Meltcurve

A melt curve (or melting curve) is a graphical representation used to analyze the thermal stability of a substance, most commonly double-stranded nucleic acids after polymerase chain reaction (PCR). In molecular biology, the melt curve is generated by gradually heating a DNA sample and monitoring a signal that reports on duplex integrity, such as fluorescence from a DNA-binding dye.

In practice, a melt curve is obtained after amplification in real-time PCR. A dye that binds preferentially to double-stranded DNA produces high fluorescence when the DNA is intact and lower fluorescence as the strands separate with increasing temperature. Plotting fluorescence versus temperature yields a curve that can be further analyzed by plotting the negative derivative of fluorescence with respect to temperature (-dF/dT). The resulting derivative peak centers at the melting temperature (Tm), the temperature at which half of the DNA duplexes are denatured. The Tm depends on sequence features such as length, GC content, and the presence of mismatches or impurities.

Melt-curve analysis is used primarily for quality control and product identification. It helps distinguish specific amplification from non-specific products or primer-dimers, assess amplicon purity, and, in some contexts, differentiate sequence variants or genotypes based on small Tm differences. The method is fast, label-free (with suitable dyes), and integrates with standard qPCR workflows.

Limitations include dependence on dye chemistry and instrument sensitivity, potential overlap of Tm values for similar sequences, the influence of salt and DNA concentration, and the need for careful baseline and data interpretation. While most common in nucleic acid workflows, the concept of melting behavior also appears in materials science, where melting curves track phase transitions in polymers and crystalline substances.