Biomedical Engineering Reference
In-Depth Information
orientated so that DNA synthesis by the polymerase
proceeds through the region between the two
primers. The extension reactions create two double-
stranded target regions, each of which can again be
denatured ready for a second cycle of hybridization
and extension. The third cycle produces two double-
stranded molecules that comprise precisely the
target region in double-stranded form. By repeated
cycles of heat denaturation, primer hybridization
and extension, there follows a rapid exponential
accumulation of the specific target fragment of
DNA. After 22 cycles, an amplification of about 10
6
-
fold is expected (Fig. 2.8), and amplifications of this
order are actually attained in practice.
In the original description of the PCR method
(Mullis & Faloona 1987, Saiki
et al
. 1988, Mullis
1990), Klenow DNA polymerase was used and,
because of the heat-denaturation step, fresh enzyme
had to be added during each cycle. A breakthrough
came with the introduction of
Taq
DNA polymerase
(Lawyer
et al
. 1989) from the thermophilic bacterium
Thermus aquaticus.
The
Taq
DNA polymerase is
resistant to high temperatures and so does not need
to be replenished during the PCR (Erlich
et al
. 1988,
Sakai
et al
. 1988). Furthermore, by enabling the
extension reaction to be performed at higher tem-
peratures, the specificity of the primer annealing is
not compromised. As a consequence of employing
the heat-resistant enzyme, the PCR could be auto-
mated very simply by placing the assembled reaction
in a heating block with a suitable thermal cycling
programme (see Box 2.3).
Cycle 1
5'+
3' Double stranded
3'-
5' DNA target
Denaturation by
heat followed by
primer annealing
5'+
3'
3'
5'
and
5' 3'
3'-
5'
DNA synthesis
(primer extension)
5'
3'
3'
5'
and
5'
3'
3'
5'
Denaturation by heat followed by primer
annealing and DNA synthesis
Cycle 2
5'
3'
3'
5'
+
5'
3'
3'
5'
+
5'
3'
3'
5'
+
5'
3'
3'
5'
Denaturation by heat followed by primer
annealing and DNA synthesis
Cycle 3
5'
3'
3'
5'
5'
3'
3'
5'
5'
3'
3'
5'
5'
3'
3'
5'
5'
3'
3'
5'
5'
3'
Fig. 2.7
(
left
) The polymerase chain reaction. In cycle 1 two
primers anneal to denatured DNA at opposite sides of the
target region, and are extended by DNA polymerase to give
new strands of variable length. In cycle 2, the original strands
and the new strands from cycle 1 are separated, yielding a
total of four primer sites with which primers anneal. The
primers that are hybridized to the new strands from cycle 1
are extended by polymerase as far as the end of the template,
leading to a precise copy of the target region. In cycle 3,
double-stranded DNA molecules are produced (highlighted in
colour) that are precisely identical to the target region.
Further cycles lead to exponential doubling of the target
region. The original DNA strands and the variably extended
strands become negligible after the exponential increase of
target fragments.
3'
5'
5'
3'
3'
5'
5'
3'
3'
5'
Repeated cycles lead to exponential
doubling of the target sequence
