View - DSpace UniPR

Chapter 2
fluorescence contribution due to the non hybridized beacon from the specific signal given by
the hybrid formation, is to use the beacon in HPLC, where the two species may be
chromatographically separated. Moreover, under the conditions in which HPLC analyses are
performed, a partial denaturation of duplexes occurs, which has been demonstrated to be
optimal for maximizing the selectivity. In particular, anion exchange HPLC has been
demonstrated to be the optimal technique to separate PNA-DNA duplexes from free DNA and
PNA. The eluents used in this method are at a slightly basic pH, which maximizes the
fluorescence intensity of fluorescein. Moreover, the ionic strength stabilize the closed
conformation of the unbound PNA-beacon, minimizing the aspecific fluorescence signal. The
only species that could interfere with the detection is the free probe, but if this molecule is
well separated from the peak corresponding to the duplex, there is no risk of interference from
other molecules, since free DNA or residues from the PCR mixture are not fluorescent. This
behavior allows to develop fast gradients. The performances in the HPLC system of the
different PNA probes with different lengths were studied in the present work.
Hybrids with fullmatched and mismatched DNA oligonucleotides having the same length as
the probe were tested and analyzed by anion exchange HPLC, using a TSK gel DNA-NPR
column. The analyses were performed with a gradient from Tris 0.02M (pH= 9) to Tris
0.02M, NaCl 1M (pH=9). The chromatograms were obtained by using a fluorescence detector
(λex= 497nm; λem= 520nm), as reported in previous works 25 . For each probe, the duplexes
were prepared in a 1:1 ratio with the fullmatch DNA, and the concentration was decreased
until it was possible to observe a clear chromatographic peak, in order to find the lowest
concentration detectable. At these concentrations, selectivity experiments were carried out in
the optimized conditions; for this reason, fullmatch and mismatch hybrids were prepared and
analyzed as previously described.
The chromatograms obtained with 11 mer and 13 mer probes showed a clear and sharp peak
corresponding to the PNA-DNA hybrids (Figure 2-6), when hybrids are formed using
fullmatch DNA. The 11 mer probe showed a good sensitivity, since the duplex could be
clearly detected down to a concentration of 30 nM (Figure 2-6 A). A broad peak eluting
before the duplex was also present: this signal can be attributed to the free probe and had not a
constant intensity, probably due to small changes in the eluent. When the 13-mer probe was
used, none of these broad signal could be detected (Figure 2-6 C), probably on account of the
higher stability of the corresponding PNA-DNA duplex. The experiments performed using
decreasing concentrations of probes and target sequences showed that the probe stabilities and
the sensitivity offered by the fluorescence detector allow to use very small amounts of probes.
46