Melting curve

Melting curve analysis is used to assess the specificity of the qPCR assay when using dyes, such as SYBR^®Green.

The melting curve analysis consists of an extra step at the end of the qPCR run. The temperature of the sample is increased incrementally while the instrument continues to measure fluorescence. As the temperature increases, double stranded DNA denatures, becoming single-stranded, and the dye dissociates, resulting in decreasing fluorescence. The change in slope of this curve is then plotted as a function of temperature to obtain the melting curve. The melting temperature of your amplicon is given by the length of the sequence and its base composition. A specific assay should optimally show a single melting peak corresponding to one, specific PCR product (see Figure 1). The melting curve analysis allows you to see if there is a primer-dimer in your qPCR, as indicated by the peak at the left side of the main amplicon peak in Figure 2. A peak after the main peak, also observed in Figure 2, could indicate unspecific amplification (e.g. the genomic sequence of this gene containing an intron, or a similar gene belonging to the same gene family, etc).

Figure 1. Ideal melting curve

Figure 2. Melting curve with primer-dimer and contamination with a longer DNA fragment.

Melting curve analysis is an important verification for the analysis of your qPCR data.