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PNA Molecular Beacons
the affinities towards oligonucleotides, the 15-mer
higher stability values.
PNA presented quite
different and much
Figure 2-5: Selectivity of PNA molecular beacons at different temperatures measured by fluorescence
spectroscopy. Selectivity has been obtained calculating the ratio between emitted
fluorescence at 522
nm of fullmatch and mismatch hybrid with excitation wavelength at 497nm.
Discrimination properties were tested in solution by
evaluating
the fluorescence switching on
in the presence of fullmatch and mismatch oligonucleotides for
a 1µM solution in Tris buffer
at pH= 8. The results shown
in figure 2-5 were obtained by
measuring the fluorescence
emission at 522 nm
for fullmatched and mismatched
hybrids and calculating the ratio between
the two
values at different temperatures.
In general, all probes
show optimal discrimination
above room temperature, suggesting that recognition experiments should
be carried out in
partially
denaturing conditions. As expected, the optimumm temperature for the probe
performance differs for the three PNAs according to the probe length: the longer the PNA, the
higher the optimumm temperature. In particular, the 15-mer probe could discriminate the point
mutation with reasonably good performances only at high temperature, confirming that this
probe is
not ideal for analytical purposes,
while smaller probes seemed to be more efficient in
the mismatch recognition especially at lower temperatures.
Comparing
the performances
offered by the two
shorter probes, it appeared that the 13 mer probe had a slightly better
selectivity in the conditions of
analysis.
A drawback of the
method emerged from
the consideration that the probe
in the closed form
had a residual fluorescence that decreases the selectivity. A way to separate the aspecific
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