504 Chapter 29
on nested PCR are available in the bibliogra-
phy (see Table 29.1 ).Quantitative PCR
In certain circumstances, such as the determi-
nation of percentage of transgenic ingredients
in a food sample, quantitative data are
required, which cannot be provided using
conventional PCR. This has been addressed,
especially using the real - time (RTi - ) PCR that
allows the quantifi cation of the initial amount
of the template by tracking the reaction cycle -
by - cycle. Another quantitative approach,
competitive PCR, can be used when the PCR
product from the sample is compared with
internal standards of known concentrations.
It coamplifi es the target sequence and one
competitor sequence (so - called internal stan-
dard) in a single reaction using conventional
PCR. Several methods have been published
using this approach for GMO quantifi cation
(Studer et al. 1998 ; Hardegger et al. 1999 ;
Zimmermann et al. 2000 ).Real - Time PCR
The principal characteristic of the RTi - PCR
is that it allows the real - time monitorization
of the synthesis of new amplicons throughout
the PCR via the fl uorescence emitted, which
is proportional to the amount of new PCR
products generated (Heid et al. 1996 ).
Therefore, the RTi - PCR results consist of
amplifi cation curves that can be used to
quantify the initial amounts of template DNA
molecules with high accuracy over a wide
range of concentrations (Schmittgen et al.
2000 ). Other advantages are the closed - tube
format, which reduces the risks of carryover
contamination, the fast and simple perfor-
mance, the wide dynamic range of quantifi -
cation (higher than six orders of magnitude),
and the signifi cantly higher reliability of the
results compared with conventional PCR.
There are two different strategies for the
detection of the new amplicon being gener-electrophoresis, by means of the ratio of
UV spectrophotometry at 260/280 nm (desir-
ably close to 1.8) and 260/230 (above 1.8 –
2.2); and by the determination of the presence
of PCR inhibitors by spiking a control
sample.
PCR Detection
Polymerase chain reaction (PCR) is a
simple, versatile, sensitive, specifi c, and
reproducible technique (Saiki et al. 1988 ). It
is an in vitro exponential amplifi cation of a
DNA fragment, and its principle is similar
to the mechanism of DNA replication. The
double - stranded DNA is fi rst denatured, and
the two strands of single - stranded DNA are
duplicated using PCR primers that specifi -
cally anneal to these strands and are elon-
gated through the activity of DNA polymerase.
This is repeated during generally 30 to 50
cycles along the reaction. Usually, the ana-
lytical result obtained by PCR is the determi-
nation of the amplifi ed products at the end
point of the reaction, especially when only
qualitative data is required. There are several
types.
Nested PCR
As conventional PCR based on the agarose
gel electrophoresis is generally less sensitive,
a modifi cation, the nested PCR, which dra-
matically increases the sensitivity and speci-
fi city of DNA amplifi cation, can be used. It
consists of two rounds of PCRs, where the
primers of the second PCR bind to an internal
region of the fi rst amplifi ed DNA product.
Thus, the larger fragment produced by the
fi rst round of PCR is used as the template for
the second round. The specifi city is particu-
larly enhanced because this technique helps
to eliminate any spurious nonspecifi c ampli-
fi cation product. However, the high risk of
cross contamination is the main drawback,
and great care must be taken when perform-
ing such PCRs. Several GMO methods based