2009_Book_FoodChemistry

2.6 Enzymatic Analysis 143
of special interest that PCR allows the detection
of genetically modified food (genetically modified
organism, GMO). Thus, it is possible to control
the labeling of GMOs, which is required by
law. In fact, the number of GMOs among food
crops is increasing steadily (cf. Table 2.19); cf.
survey by Anklam et al. (2002).
2.6.4.1 Principle of PCR
The first steps of a PCR reaction are shown
schematically in Fig. 2.45. First, the extract
which contains, among other substances, the
DNA fragment (analyte) to be identified is briefly
heated to 95 ◦ C. This causes denaturation and
separation into single strands. After cooling
to 54 ◦ C, two oligodeoxynucleotides (primer 1
and 2 having base sequences complementary to
the target DNA) which flank the DNA sequence
to be multiplied are added in excess. These
primers, which are 15–30 nucleotides long and
made with a synthesizer, hybridize with the complementary
segments on the single strands. The
temperature is increased to 72 ◦ C and a mixture
of the four deoxynucleoside 5 ′ -triphosphates
(dATP, dCTP, dGTP, dTTP, for structures of the
bases cf. Formula 2.107) and a thermostable
DNA polymerse, e. g., Taq polymerase from
Thermus aquaticus, are added. The polymerase
synthesizes new complementary strands starting
from the primers in the 5 ′ → 3 ′ direction using the
deoxynucleotides. In the subsequent heating step,
these strands are separated, in addition to the
denatured target DNA which is no longer shown
in Fig. 2.45. In the second cycle, the primers
hybridize with the single strands which end with
the nucleotide sequence of the other primer in
each case. The PCR yields two DNA segments
(a and b in Fig. 2.45) which are bounded by
the nucleotide sequences of the primer. The
DNA segment is amplified by repeating the steps
denaturation – addition of primer – PCR 20 to
30 times, and is then electophoretically analyzed.
(2.107)
Fig. 2.45. Principle of PCR
In comparison with protein analysis, DNA analysis
is more sensitive by several orders of mag-