View - DSpace UniPR

Chapter 2
lower sensitivity, and significant peaks could be obtained only by using relatively higher
concentrations (200nM, Figure 2-7). Finally, although some good chromatograms were
obtained, a high variability in the peak intensity was noticed, making the probe not
particularly suitable for this assay. To make bad things worse, a PNA-DNA hybrid could be
clearly detected also when the beacon was hybridized with the mismatched oligonucleotides.
Figure 2-7: Chromatogram obtained for fullmatched and mismatched hybrids using PNA Molecular
Beacon 15 mer 200nM at different temperatures. The analytical conditions are the same, as described
above
Since the only difference between this PNA and the other previously reported is the length,
these problems should be related to this parameter. The increased length may cause selfaggregation
in solution, due to the increased hydrophobicity, and a slower rate in the
hybridization process also with complementary sequences. Moreover, the 15-mer beacon is
less selective on account of the higher stability of the fullmatch and mismatch PNA-DNA
hybrids at room temperature. In order to completely denaturate the mismatched duplex, the
column temperature was increased. The chromatograms in figure 2-7 show that warming up
to 35°C was necessary in order to get rid of the aspecific signal, but at this temperature the
fullmatch hybrid was also highly destabilized, as demonstrated by its strongly decreased
signal.
Since all the results suggested that the optimal probe, in terms of selectivity and sensitivity,
was a PNA beacon 13 bases long, this probe was used in the optimized conditions for the
recognition of the DNA samples extracted from olive leaves and amplified by PCR. DNA
extracted from leaves of two different cultivars of olive were used: one from “Ogliarola
Leccese”, bearing the SNP at position 60 (thymine versus adenine) of the actine gene, for
which the probe has been designed; the other from “Canino”, which was chosen as negative
control, since it has an adenine at position 60 as other eleven cultivars. The extracted DNA
was amplified by Polymerase Chain Reaction (PCR) and purified with an affinity column.
48