112
and as the Tm is characteristic of the GC content, length and sequence of a DNA product, it is a
useful tool in product identification.
Dissociation curve showing the dissociation
peaks.
At the start of an analysis, the reaction is at a low temperature such that the DNA in the reaction
tube will be double-stranded and the fluorescence signal high. As the temperature slowly ramps
upwards, the fluorescence will drop more rapidly as the reaction reaches the melting point and the
dye can no longer intercalate. Pure, homogenous PCR products will produce a single sharply
defined melting curve with a sharp peak, while primer-dimers will melt at a lower temperature with
a broader peak.
Dissociation curve analysis has applications in such areas as genotyping and mutation analysis,
PCR optimization and the identification and characterization of PCR products.
Tem perature
92
90
88
86
84
82
-d
F
/d
T
2,500
2,000
1,500
1,000
500
0
