Assessment of Genetically Modifi ed Organisms (GMO) in Meat Products by PCR 503However, particle size, composition, or heat
and pressure processing might induce distor-
tions in the results of GMO quantifi cation
due to their infl uence on DNA degradation
(Moreano et al. 2005 ; Hird et al. 2006 ). Then,
cell membrane lysis by enzymatic activity
and/or mechanical disruption is performed,
mostly in the presence of detergents (guani-
dinium isothiocyanate, SDS, CTAB) and
chelating agents (EDTA); cleanup steps
come next, using organic solvents (chloro-
form, phenol); and fi nally, DNA is separated
and concentrated by alcohol/salt precipita-
tion (ethanol or isopropanol), affi nity, or ion/
exchange purifi cation columns (Hernandez et
al. 2005 ). One of the most commonly used
procedures for DNA extraction in GMO
analysis is the CTAB - based protocol (Rogers
and Bendich 1985 ) or its variations (Meyer
and Jaccaud 1997 ). Binding and elution from
silica has also become the procedure of
choice for most nucleic acid extraction
procedures (Smith et al. 2003 ). In addition,
many different commercial kits and auto-
mated procedures for food samples are cur-
rently available (Marmiroli et al. 2008 ),
although the automation is restricted nowa-
days to a limited number of food samples
(Hahnen et al. 2002 ). Several papers compare
different extraction methods (Zimmermann
et al. 1998a ; Peano et al. 2004 ).
After DNA extraction, quantity and
quality must be evaluated by means of UV
spectrophotometry at 260 nm; by fl uorimetry
using fl uorescent ds - DNA - specifi c dyes; or
by visualization under UV light of DNA
separated in agarose gels stained with ethid-
ium bromide. Before DNA estimation, RNA
must be carefully eliminated in order to avoid
an overestimation of yield. For this purpose
a straightforward treatment with RNAase A
is recommended. Then, it is simple to cor-
relate the amount of DNA (ng) with copy
number by using the C - value (Arumuganathan
and Earle 1991 ; Bennett and Leitch 2003 ).
Purity of DNA can be evaluated by assessing
the degree of degradation using agaroseodological approach focused on food safety,
especially in GMO analysis, is the selection
of a sound and rational sampling strategy
(Michelini et al. 2008 ). The sample taken for
analysis must be statistically representative
of the original population or batch. This issue
is of particular importance for validation
studies where protocol performance is
assessed. Intimately related with this is the
selection of the correct weight or volume that
must be processed from each sample.
Generally, a portion about 0.1 to 1 g from the
received sample is considered suffi cient for
processing (Pietsch et al. 1997 ; Zimmermann
et al. 1998a, b, c ; van Duijn et al. 2002 ).
The DNA - based methods, especially
those based on its amplifi cation, rely on dif-
ferent analytical steps: the DNA extraction,
the DNA amplifi cation by PCR, and fi nally,
the detection of the specifi c amplifi cation
products. In the case of the use of real - time
PCR - based methods, the two later steps can
be combined into a single one, as the ampli-
fi cation and detection occurs simultaneously
in a single analytical step.
DNA Extraction from Food Matrices
The fi rst step of PCR - based methods in
food analysis relies on a careful DNA -
extraction procedure, since components of
food samples and nucleic acid extraction
reagents can reduce or even block the PCR
amplifi cation, and DNA may be degraded.
Food components that can act as PCR inhibi-
tors are proteins, fatty acids, and other sec-
ondary compounds such as polyphenols.
Consequently, adequate nucleic acid purifi -
cation is crucial to convert food samples into
samples amenable for amplifi cation by adap-
tation of extraction procedures to each food
matrix. The fi rst stage is homogenization to
reduce the sample particles to an appropriate
size by grinding in homogenizers, such as
blenders, stomacher, polytron, ultra - turrax,
mills, and mortars, which reduce consider-
ably the sampling error (Begg et al. 2007 ).