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Chapter 5
22.4°C 24.0°C 25.5°C 27.2°C
29.0°C 30.8°C 32.7°C 35.3°C
36.8°C 38.2°C 39.6°C 41.7°C
42.2°C
Figure 5-7: Images of fullmatch TAMRA-labeled PNA-DNA duplex at different temperatures.
Images were taken using a laser scanning confocal microscope
The fluorescence intensity was measured in three different areas of the picture, and then
averaged; at the same time, the same was done for the background, which was subtracted
from the fluorescence intensity. These averaged and normalized values were plot against the
temperature, resulting in a sigmoid curve typical of the cooperative behavior shown by PNA-
DNA duplexes during their melting (Figure 5-8). The possibility to fit the points with a
sigmoidal curve allowed to calculate the flex point, obtaining the melting temperatures for
these hybrids directly on surface. In table 5-2 the values obtained on surface are compared
with the melting temperatures in solution, either measured in PBS or in SSC 2x SDS 0.1%,
order to have the same buffer used for the hybridization on surface. It is possible to notice that
a significative destabilization both of the fullmatch and of the mismatch PNA-DNA duplexes
in solution was caused by the presence of SDS; indeed this surfactant, being negatively
charged, apparently interferes with the positively charged PNAs in DNA binding, decreasing
the stability of the duplexes of about 10°C (Tm from 55°C to 45°C for the fullmatch duplex
and from 35° to about 25°C for the mismatch duplex). The data obtained on the surface show
that, for fullmatch PNA-DNA duplexes, a further destabilization was introduced when the
binding took place between molecules in different phases, with the Tm decreasing from 45°C
100