FAQ: Why do I have multiple peaks in my melt curve?

A typical denaturation (melt) curve performed after qPCR cycling with an intercalating dye, will typically give rise to a single distinct peak in the plot of the negative derivative of fluorescence vs. temperature. This indicates that the amplified double-stranded DNA products are a single discrete species. The presence of multiple DNA species in the same reaction can give rise to multiple peaks in the melt curve, typically indicating the presence of contaminating or off-target amplification products. Search the PCR primers against the input DNA sequence to determine if a product other than the desired target can be amplified, for example, due to sequence similarity or multiple forms of the same gene sequence. Redesign primers if necessary. Occasionally, due to sequence-specific characteristics and/or GC content, certain qPCR products will experience multi-stage melting transitions, giving rise to a melt curve with multiple peaks. These types of products can be treated as a successful qPCR. The melt profile of any sequence of interest can be predicted using the online tool uMelt.