View - DSpace UniPR

PNAs: from birth to adulthood
DNA strand labeled with a group that can be recognized by an enzyme. The enzyme
recognition starts a series of reactions that eventually produce a fluorescence signal 101 .
PCR-free techniques are the very ultimate goal and a very difficult challenge for scientists
that work in the field of DNA recognition, so that many efforts have been put in the
realization of systems able to perform such assays. One of these systems is based on Surface
Plasmon Resonance (SPR), a phenomenon which induces a change in the refractive index due
to the increase of mass on the surface. If a PNA probe is linked to the surface, hybridization
with the DNA/RNA complementary sequences will increase the mass laying on the surface
giving rise to an SPR signal. If the target DNA is labeled with bulky groups (such as gold
nanoparticles), or a sandwich assay is performed, using a further probe linked to gold
nanoparticles, this method can become very sensitive, thus allowing the detection of DNA
without the necessity of pre-amplification 102 . This technique has been applied using a 15-mer
PNA sequence specifically designed to identify Roundup Ready (RR) genetically modified
soybean, obtaining a selective sequence discrimination with a femtomolar sensitivity 102 .
Sandwich assays like the ones described above have been used for label-free DNA detection,
exploiting the hybridization of the target sequences with the PNA probe on one side and with
a revealing labeled PNA probe on the other side.
Also other interactions have been exploited in order to form such sandwiches for introducing
different types of labeling groups. The interaction between a DNA strand hybridized to the
PNA and a Zr 4+ ion was used for introducing a rhodamine label that interact with the ion that
is coordinated by DNA phosphates 103 . This fluorophore can be detected by Surface Enhanced
Raman Scattering techniques on gold and silver derivatised silicon surfaces.
Finally, QCM microbalance is another technique that can be exploited to detect non labeled
nucleic acids, since the signal is given by the increase of mass linked to the sensor, which
increases when a DNA strand hybridizes with the PNA probe 104,105 .
Another critical point in the analysis of nucleic acid sequences is the matrix interferences
found in the biological samples. DNA extraction and purification from a real sample is not
always an easy step, and for this reason PNAs linked to surfaces have been applied in DNA
purification by hybridization. PNAs, in fact, can be used to complex DNA molecules in
complex matrices, separating them from other interferents. In this way, a concentration effect
can be achieved, and DNA can be purified in a suitable way for subsequent PCR
amplification and detection 106 .
DNA extraction is not always necessary for diagnostic applications in vivo, since sometimes
hybridization can be performed directly in the cells. Fluorescent PNA oligomers labeled with
33