34 Landgraf and Wolfes- Polymerase Chain Reaction (PCR)
The first publication concerning the polymerase chain reaction (PCR)
appeared in 1985 (2). In this protocol, Klenow polymerase was used
as the replicating enzyme, thus necessitating the addition of new poly-
merase after each denaturation step. In 1988, the first use of Taq poly-
merase for the PCR was reported (3), leading the way to an automation
of the protocol. The application of Taq polymerase to the PCR was the
basis for the success of the technique. The enzyme is extremely suit-
able for the following reasons:
- Taq polymerase is stable up to 95°C; thus it is not necessary to replen-
ish the enzyme after each PCR cycle; - The maximal enzyme activity is between the temperature range of 70-
75°C, which minimizes secondary structures of the template, resulting
in high polymerization yield; and - The annealing temperature can be chosen from 30-70°C, allowing an
optimal adaptation of cycle parameters to appropriate annealing tem-
peratures of the primers; therefore, byproducts are hardly generated.
3.1. Fundamentals of the PCR
The PCR is an enzymatic chain reaction that leads to the amplifica-
tion of specific DNA sequences from a given template (11). In addition
to the template, two primers (typically: oligodeoxynucleotides of 20
nucleotides in length) are needed, whose sequence must be comple-
mentary to the template DNA. The amplification is performed in the
presence of dNTPs as substrate and catalyzed by Taq polymerase. The
sequence between the two regions complementary to the primers is
doubled by repeating the cycle, which consists of a series of tempera-
ture steps in which the DNA is denatured, the primers are annealed,
and the polymerization of the specific sequence occurs. The newly
synthesized sequence is available as template in the next cycle; there-
fore, theoretically, a twofold increase of template is achieved in each
step. In theory, 30 cycles should yield an amplification of a specific
sequence by a factor of 230= 1 × 109, as long as substrates or enzyme
is not limiting (Fig. 1).
The PCR technique reaches outstanding results because of the fact
that the chain reaction requires the specific annealing of two primers
to a complementary sequence. Unspecific annealing of primers or
nicks in the template does not lead to exponential, but only linear