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Chapter 1
1.8.1 Analytical Applications
The recognition of a DNA sequence in a biological sample usually takes place in three steps:
the extraction and purification of the DNA from the sample, the amplification by PCR of the
genomic region containing the sequence to be detected, and the recognition of the sequence.
Although DNA sequencing is often possible, the necessity to develop new assays based on
probes that rapidly recognize a specific sequence is also necessary, because the sequencing is
time-consuming, expensive and often made impossible by the low amounts of DNA. The use
of specific probes can make the recognition more robust, faster and cheaper. PNAs are
particularly suitable for these applications, because of their chemical stability, the affinity for
DNA which enhances the sensitivity, the possibility of forming duplexes at low ionic strength
that allows the hybridization in conditions where the DNA does not form secondary
structures, and the selectivity, especially in the recognition of point mutations.
DNA detection can be performed in solution or on surface. In solution assays, the PNA
probes and the DNA to be analyzed are in homogeneous solution; a great number of different
assays have been developed, being based on different readout signals. The easiest signal that
can be exploited is the change of color of the sample, in colorimetric assays, when the this
happens upon the nucleic acid hybridization. Molecules such as cyanine DiSC 2 (5) dye, able to
interact with PNA-DNA double helix but not with PNA or DNA single strands, giving an
ipsochromic effect due to aggregation which result in an instantaneous color change, have
been exploited for these kinds of essays 85 . A colorimetric test using Disc2(5) dye, PNA and
Succ-β-CyD was successfully applied for the detection of SNPs correlated to the
apolipoprotein E gene, which have been linked to the Alzheimer disease 86 . Another effect
causing changes in the color of a solution is the aggregation state of nanoparticles; the use of
PNAs with gold nanoparticles was demonstrated to cause their collapse, that is reversible
when complementary DNA is added, causing a different absorption band 87 .
The possibility to link the PNA to a superparamagnetic iron oxide nanoparticles (MNP)
obtaining MNP-conjugated PNAs (MPNA). These nanoparticles have been used for the
detection of complementary DNA sequences, exploiting their capability of MPNA to enhance
the relaxation time T2 in aqueous solutions when specific hybridization takes place 88 .
More sensitive assays are those based on fluorescence, by exploiting the appearance of a
signal upon PNA-DNA duplex formation. These particular PNAs are designed to be non
fluorescent when non hybridized, and to have a conformational change when the
hybridization takes place, leading to the lighting up of a fluorescent signal. PNA molecular
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