The PCR
(Polymerase Chain Reaction)
is an in vitro method for the
enzymatic synthesis of specific DNA sequences, using two oligonucleotide
primers that hybridize to opposite strands and flank the region of interest in
the target DNA. A repetitive series of cycles involving
template denaturation , primer annealing, and the extension of the annealed
primers by DNA polymerase results in the exponential accumulation of a specific
fragment whose termini are defined by the 5' ends of the primers. Because the
primer extension products synthesized in one cycle can serve as a template in
the next, the number of target DNA copies approximately doubles at every cycle.
Thus, 20 cycles of PCR yields about a million-fold ( 
) amplification.
This method, which was invented by Kary
Mullis [84, 85] was
originally applied by a group in the Human Genetics Department at Cetus to the
amplification of human 
-globin DNA and to the prenatal diagnosis of
sickle-cell anemia[86, 87, 88].
Initially, the PCR used the Klenow fragment of E. coli DNA
polymerase I[84, 86] to extend the annealed primers. This enzyme was inactivated by the
high temperature required to separate the two DNA strands at the outset of each
PCR cycle. The introduction of the thermostable DNA polymerase
(Taq polymerase[89])
isolated from Thermus aquaticus transformed the PCR into a
simple and
robust reaction which could now be automated by a thermal cycling device.
The effect of varying the reaction parameters (e.g., enzyme,
primer and Mg 
concentration as well as the temperature cycling
protocol) is discussed below. Although, for any given pair of oligonucleotide
primers, an optimal set of conditions can be established, there is no single
set of conditions that will be optimal for all possible reactions.
The initial PCR method based on DNA synthesis by the
Klenow enzyme at 37psy176
C was not highly specific. The use of the Taq polymerase not only
simplified the PCR procedure but significantly increased the specifity and the
overall yield of the reaction. The higher temperature optimum for the
Taq polymerase
( 
75psy176 C) allowed the use of higher
temperatures for primer annealing and
extension, thereby increasing the overall stringency of the reaction and
minimizing the extension of primers that were mismatched with the template.
Although the PCR is considered primarily a method for producing copies of a
specific sequence, it is also a very powerful and precise way of altering a
particular template sequence. Since the oligonucleotide primers become
physically incorporated into the amplified product and mismatches between the
5' end of the primer and initial template are tolerated, it is possible to
introduce new sequence information adjacent to the target sequence via the
primers. Thus, for cloning a given sequence, one is no longer constrained by
the naturally occurring restriction sites and one may add any restriction enzyme
recognition sequence to the 5' ends of the primer[91] creating a
new restriction site in the double-stranded amplification
product. Furthermore, specific nucleotide substitutions, insertions, and
deletions can also be introduced into the amplified product with the
appropriate primers.