Assessment of Genetically Modifi ed Organisms (GMO) in Meat Products by PCR 507FRET hybridization probes. Among the dif-
ferent types of probes currently available in
the market, the TaqMan probes are nowadays
the probes of choice.
The fl uorescence is determined cycle - by -
cycle by the RTi - PCR platform, and the
result is an amplifi cation plot. A typical
amplifi cation curve presents three different
phases. The initiation phase occurs during
the fi rst PCR cycles when the emitted fl uo-
rescence cannot be distinguished from the
baseline. During the exponential or log phase
there is an exponential increase in fl uores-
cence, and fi nally, during the plateau phase,
the reagents are exhausted, and no increase
in fl uorescence is observed. Quantifi cation is
only possible, therefore, at the beginning of
the exponential phase when the reaction
is totally effi cient and all the reagents are
available. The most important parameter in
the RTi - PCR is the threshold cycle (C T value)
(Higuchi et al. 1992 ), which is used for the
quantifi cation of the sample. It corresponds
to the cycle at which a statistically signifi cant
increase in amplifi cation - associated fl ores-
cence is fi rst detected, and is inversely cor-
related to the concentration of DNA present
in the original sample (Walker 2002 ). The
initial DNA concentration of the sample
can then be determined by interpolation
of the resulting C T value in a linear standard
curve of serially diluted, known - amount
standards.Other DNA Techniques
Other modifi cations and techniques based on
DNA detection are continuously appearing in
the scientifi c literature (Garcia - Canas et al.
2002 ; Burns et al. 2003 ; Feriotto et al. 2003 ;
Rudi et al. 2003 ; Glynou et al. 2004 ; Obeid
et al. 2004 ; Fantozzi et al. 2008 ; Morisset et
al. 2008 ). Recently, a novel DNA - based
technology has been developed, the NASBA
Implemented Microarray Analysis (NAIMA)
(Morisset et al. 2008 ). In this technology, a
cRNA product is obtained from a consecu-probe or specifi c DNA polymerases. However,
the specifi city is determined entirely by the
primers, as in conventional PCR, and thus the
risk of amplifying nonspecifi c PCR products
increases (Simpson et al. 2000 ). It is possible
to verify the correct production of a given
PCR product by means of plotting fl uores-
cence as a function of temperature to generate
a melting curve of the amplicon at the end
point (Ririe et al. 1997 ). Another nonspecifi c
detection system is the Sunrise primers
(AmpliFluor^ ™^ system) developed by Intergen
Co. It uses a universal, energy - transfer hairpin
primer (UniPrimer^ ™^ ) that emits a fl uorescent
signal when unfolded during its incorporation
into an amplifi cation product (Nazarenko
et al. 1997 ).
The “ sequence - specifi c fl uorescent
probes ” can be classifi ed into two major
groups: hydrolysis and hybridization probes.
Both types consist of an oligonucleotide,
homologous to the internal region of the
amplicon, that is double - labeled, with a fl uo-
rophore or reporter dye (donor of fl uores-
cence) at the 5 ′ - end and a quenching moiety
(acceptor of fl uorescence) at the 3 ′ - end. The
distance between the fl uorophores is a key
factor in generating sequence - specifi c signals
(F ö rster 1948 ; Clegg 1992 ), and a change in
the distance between them is used to produce
the RTi - PCR signals. The hydrolysis probes
such as TaqMan ® probes and TaqMan ® MGB
probes are cleaved by the 5 ′ - 3 ′ exonuclease
activity of several DNA polymerases during
the primers ’ elongation phase (Holland et al.
1991 ), yielding a real - time, measurable fl uo-
rescence emission directly proportional to the
concentration of the target sequence. In con-
trast, hybridization probes are not hydrolyzed
during PCR. The fl uorescence is generated
by a change in its secondary structure during
the hybridization phase, which results in an
increase of the distance that separates the
reporter fl uorophore from the quencher
moiety. The most relevant hybridization
probes are those containing hairpins such as
Molecular Beacons, Scorpion primers, and