Melting Curve Analysis in qPCR
Researchers often use melt curve analysis to assess whether their intercalating dye PCR/qPCR assays have produced single, specific products. Specificity of amplification is considered to be a greater concern with intercalating dye assays than with probe-based assays because intercalating dyes bind to any double stranded DNA product and are not sequence specific. With probe-based assays, amplicon specific primers gain an additional level of specificity from the probe, which is a third sequence that has to bind within the amplicon of the target sequence in order to generate a fluorescent signal.
Melt curve analysis is frequently used as a diagnostic tool for assessing qPCR amplicon length with intercalating dye qPCR assays. Here, we explain how melt curves are produced, examine the assumptions being used, and describe some additional methods that can be used to further analyze melt curve results.
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