Melting Curves Real Time PCR

Melting Curves Real Time PCR

Real Time PCR Melting Curves

Melt curves require the presence of an intercalating agent and can be performed on qPCR runs which have used SYBR green to detect amplification or by adding SYBR green to the post qPCR tubes. A melt curve is a plot of temperature (x-axis) against the rate of change of fluorescence (y-axis) following a slow ramping from 50 ° C to 90 ° C.

SYBR green fluoresces more intensely when bound to double-stranded DNA than in solution and the fluorescence will therefore decrease as the PCR products melt, releasing bound SYBR green. However, when the melting point of a PCR products is reached, the rate of decrease in florescence (y-axis) is high resulting in a peak at this temperature. A melt curve is very similar to running an agarose gel to size PCR bands although resolution is based on melting point rather than the length of the PCR product. The melt curve from a qPCR assay that amplifies on a single target will produce one sharp peak corresponding to the melting point of the target amplicon.

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