508 Chapter 29
junction regions of DNA from different
origins, and fl anking regions of the intro-
duced construction, which aims to determine
which GMO is present in the sample, and
therefore, if it is authorized or not; and
fi nally, (3) GMO - quantifi cation that targets
the specifi c gene, or the border sequences,
and accurately quantifi es the percentage of
GMO in the food product as a necessary step
for labeling if required by the norm in force
(H ü bner et al. 1999 ). In any case, DNA from
the plant species should be analyzed by using
an endogenous gene in the amplifi cation
reactions that can be used as a reference to
normalize the GMO content.Analysis of Regulatory Sequences and
Marker Genes
Screening methodologies exploit the detec-
tion of common elements present in GMOs,
such as marker genes or regulatory sequences
(i.e., promoters and terminators). Most com-
mercially approved GMOs have been trans-
formed using constructs containing sequences
from the Caulifl ower Mosaic Virus (CaMV,
i.e., 35S promoter [P - 35S] and/or 35S termi-
nator [T - 35S]) or from Agrobacterium tume-
faciens (i.e., nopaline synthetase terminator
[T - nos ]). Screening methodologies have also
traditionally used selectable marker genes
that encode proteins that confer herbicide
or antibiotic resistance (Draper and Scout
1991 ; Flavell et al. 1992 ; Kok et al. 1994 ;
MacCormick et al. 1998 ). The most accepted
marker gene has been npt II, encoding resis-
tance to aminoglicosidic antibiotics (neomi-
cin/kanamicin). The bla gene, encoding
ampicilin resistance, and the bar gene, encod-
ing phosphinothricin tolerance, have also
been widely employed (D ’ Halluin et al.
1992 ). Therefore, most of the screening
methods are based on the detection of P - 35S,
T - nos , and bla and npt II genes. Another
strategy is the use of regions from cloning
vectors regularly used for transformation
(i.e., plasmid sequences derived from pBR322tive fi rst primer extension reaction, using
tailed primers, and a subsequent transcrip-
tion - based amplifi cation, using universal
primers. The NAIMA product is directly
ligated to fl uorescent dyes labeled 3DNA
dendrimers, allowing signal amplifi cation
and hybridized without further purifi cation
on an oligonucleotide probe - based microar-
ray for multiplex detection. Another inge-
nious hybrid protocol has been published
aimed at the simultaneous quantifi cation of
multiple nucleic acid targets (Rudi et al.
2003 ). This approach, named MQDA - PCR,
is based on two - step multiplex amplifi cation
of target sequences, followed by sequence -
specifi c labeling of the probes to be used for
DNA - array hybridization. The approach has
been tested using diluted mixtures of certifi ed
GMO material, as well as with commercial
food samples with similar success. Fantozzi
et al. (2008) have developed a screening pro-
tocol for the detection of the regulatory
sequence p35S and the specifi c event epsps.
It is based on the Luminex xMAP technol-
ogy, and two different sets of fl uorescent
beads are cross - linked to the specifi c oligo-
nucleotide probes previously amplifi ed and
labeled by polymerase chain reaction (PCR)
in the presence of a biotinylated nucleotide.
However, an important aspect that must
be considered prior to routine use of these
techniques in food analysis is the validation
of their performance through use by the sci-
entifi c community as has occurred with the
real - time PCR methods.
Strategies for Detection,
Identifi cation, and Quantifi cation
of GMO s
Different analytical strategies have been
exploited in GMO analysis: (1) screening
methodology that commonly detects regula-
tory sequences or marker genes, and which
aims to detect the presence or absence of
GM - material; (2) GMO identifi cation via the
identifi cation of specifi c genes, detection of