Food Chemistry

2.6 Enzymatic Analysis 143

of special interest that PCR allows the detection
of genetically modified food (genetically modi-
fied organism, GMO). Thus, it is possible to con-
trol 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 PrincipleofPCR........................................

The first steps of a PCR reaction are shown
schematically in Fig. 2.45. First, the extract

Fig. 2.45.Principle of PCR

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 com-

plementary 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)

In comparison with protein analysis, DNA ana-

lysis is more sensitive by several orders of mag-