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Melt curve analysis may also be used for genotyping purposes based on the differences in melt
temperature between alleles.
There are two types of curves used in the melt analysis application: normalized and derivative.
Normalized Plot
: This displays the various temperature increments on the x-axis and the
normalized fluorescence signal intensity on the y-axis. Melt curve analysis function by
incremental temperature changes over time, thereby resulting in DNA strand dissociation.
Since the overall fluorescence intensity is directly proportional to the amount of double-
stranded target DNA, strand dissociation results in decreased fluorescence intensity.
The inflection point seen in the sigmoidal shaped curve below equates to the melting
temperature (T
m
) of the amplified product.
Figure 5.9
Derivative Plot
: This is a plot of the negative first derivative of the normalized curve. Each peak
is characteristic for a specific amplicon. The T
m
is easily identified here since it is the maximum
point of the peak. The T
m
is defined as the temperature at which 50% of the product has
“melted” from double-stranded DNA to single-stranded DNA.
In Figure 5.10 below, temperature is displayed on the x-axis and the negative first derivative of
the normalized curve is displayed on the y-axis (-dF/dT).
Figure 5.10
