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PNA Molecular Beacons
In particular, the 11mer probe could be efficiently set at the concentration of 30 nM (Figure 2-
6 B), while the higher stability offered by the 13mer probe allowed to work at a concentration
of 10 nM (Figure 2-6 D), reaching limits which could not be afforded in previous similar
works 21, 25 .
Figure 2-6: AE-HPLC-FD chromatograms (λex= 497nm; λem= 520nm) obtained for PNA beacon-
DNA fullmatch hybrids at various concentrations with 11-mer (A) and 13-mer (C) probes.
Chromatograms obtained with fullmatch DNA and single mismatch DNA at concentrations of 30 nM
for 11-mer (B) and 10 nM for 13-mer (D) just above the limit of detection are shown. Conditions:
TSK gel DNA-NPR column with a gradient from 100% A (Tris 0.02M, pH= 9) to 100% B (Tris
0.02M, NaCl 1M pH=9).
The lowest concentrations detectable were then used to perform selectivity tests, by using
oligonucleotides containing one mismatch exactly corresponding to the SNP characteristic of
other olive varieties. The chromatograms obtained (Figure 2-6) show that both probes did not
give rise to any peak when mismatched oligonucleotides were used, allowing to recognize the
mutation with an excellent selectivity at room temperature.
The data obtained with the 15 mer PNA beacon, exactly mirroring the results in solution,
were less clear than those with the shorter probes. The chromatograms always showed a
double peak of unknown origin corresponding to the duplex signal, that could not be
attributed to impurities or byproduct since either PNA and DNA were HPLC-purified and MS
characterized. Moreover, the recognition experiments performed with this probe showed a
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