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4.7 Analysis method: Dissociation curve
Dissociation curve analysis can add to the information obtained from the PCR. Also known as
melting curve analysis, it measures the temperature at which the DNA strands dissociate (i.e. the
melting temperature or Tm). Using an intercalating dye such as SYBR
®
Green I and increasing the
temperature in small increments, the PCR products can be seen to ‘unzip’ at a specific
temperature. Since the melting temperature is characteristic of the GC content, length and
sequence of a DNA product, it is a useful tool in product identification.
Dissociation curve analysis has applications in:
•
Identifying and characterizing PCR products.
•
Genotyping and mutation analysis using hybridization probes.
•
Optimizing PCR: If the Tms of a PCR product and primers are known, the annealing
temperature can be kept above the Tm of the primer-dimers but below the Tm of the specific
PCR product.
•
Estimating relative concentration of products: Peak area analysis can provide an indication
of the relative amount of each product.
Dissociation curve analysis is an inversion of the PCR amplification method in as much as a cycle-
to-cycle decrease in fluorescence is seen as opposed to an increase.
•
At the start of an analysis, the reaction is at a relatively low temperature and the
fluorescence signal high.
•
As the temperature slowly ramps upwards, the fluorescence will drop more rapidly as the
reaction reaches the melting point.
•
Pure, homogenous PCR products will produce a single sharply defined melting curve with a
sharp peak, while primer-dimers will generally melt at a lower temperature with a broader
peak.
Dissociation peak analysis is a plot of the negative first derivative of the dissociation curve which
shows a characteristic peak for each product (the derivative is the negative of the rate of change in
fluorescence as a fraction of temperature). By taking the derivative of the dissociation curve as
opposed to using the raw data, identifying the Tm is made easier as a peak is produced:
The Tm is the maximum point of the peak and the temperature at which 50% of the product has
melted. Peak area analysis measures the area under a peak and can provide an indication as to
the relative amounts of each product. Each analysis follows on from the next so to calculate peak
areas, for instance, it is necessary to have already analysed the dissociation peaks and Tms.
Negative first derivative
Temperature
94 92 90 88 86 84 82 80 78 76 74 72
-d
F
/
d
T
2,600
2,400
2,200
2,000
1,800
1,600
1,400
1,200
1,000
800
600
400
200
0
-200
-400
Dissociation curve
Temperature
90 85 80 75
F
lu
o
re
s
c
e
n
c
e
9,000
8,000
7,000
6,000
5,000
4,000
3,000
2,000
1,000