An introduction to PCR
1.1 Introduction: PCR, a ‘DNA photocopier’
Does it really work? It is so simple! Why did I not think of it? These
thoughts were probably typical of most molecular biologists on reading
early reports of the polymerase chain reaction or PCR as it is more
commonly called. PCR uses a few basic everyday molecular biology reagents
to make large numbers of copies of a specific DNA fragment in a test-tube.
PCR has been called a ‘DNA photocopier’. While the concept is simple, PCR
is a complicated process with many reactants. The concentration of
template DNA is initially very low but its concentration increases dramatic-
ally as the reaction proceeds and the product molecules become new
templates. Other reactants, such as dNTPs and primers, are at concentra-
tions that hardly change during the reaction, while some reactants, such
as DNA polymerase, can become limiting. There are significant changes in
temperature and pH and therefore dramatic fluctuations in the dynamics
of a range of molecular interactions. So, PCR is really a very complex
process, but one with tremendous power and versatility for DNA manipu-
lation and analysis.
In the relatively short time since its invention by Kary Mullis, PCR has
revolutionized our approach to molecular biology. The impact of PCR on
biological and medical research has been like a supercharger in an engine,
dramatically speeding the rate of progress of the study of genes and
genomes. Using PCR we can now isolate essentially any gene from any
organism. It has become a cornerstone of genome sequencing projects, used
both for determining DNA sequence data and for the subsequent study of
putative genes and their products by high throughput screening method-
ologies. Having isolated a target gene we can use PCR to tailor its sequence
to allow cloning or mutagenesis or we can establish diagnostic tests to
detect mutant forms of the gene. PCR has become a routine laboratory tech-
nique whose apparent simplicity and ease of use has allowed nonmolecular
biology labs to access the power of molecular biology. There are many

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5'
dNTPs
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like an RNA polymerase that does not require a primer then we would have
no way of defining what segment of DNA we wanted to be copied.
At the next heating step the double-stranded molecules, which are
heteroduplexes containing an original template DNA strand and a newly
synthesized DNA strand produced during the first DNA synthesis reaction,
are now denatured. Each DNA single strand can now act as a template for
the next round of DNA synthesis. As discussed in detail in Chapter 2, it is
during this second cycle of PCR that the first DNA single strand of a length
defined by the positions of the primers can be formed. In cycle 3 the first
correct length double-stranded PCR products are formed. In subsequent
cycles there is then an exponential increase in the number of copies of the
‘target’ DNA sequence; theoretically, the number of copies of the target
sequence will be doubled at each PCR cycle. This means that at 100%
efficiency, each template present at the start of the reaction would give rise
to 10
6
new strands after only 20 cycles of PCR. Of course the process is not
100% efficient, and it is usually necessary to carry out more reaction cycles,
often 25 to 40 depending upon the concentration of the initial template
DNA, its purity, the precise conditions and the application for which you
require the product. The specificity and efficiency of PCR, however, means
that very low numbers of template molecules present at the start of the PCR
can be amplified into a large amount of product DNA, often a microgram
or more, which is plenty for a range of detailed analyses. Of course, this
ability to amplify also means that if you happen to contaminate your
reaction with a few molecules of product DNA from a previous reaction, you
may get a false result. This is why performing control reactions is so impor-
tant and we will deal with such contamination problems in Chapter 4.
1.3 PCR is controlled by heating and cooling
PCR relies on the use of different temperatures for the three steps of the

Synthesis of new DNA strands defined by primers
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target DNA it is necessary to cycle through these temperatures several times
(25 to 40 depending on the application). Conveniently, this temperature
cycling is accomplished by using a thermal cycler, a programmable instru-
ment that can rapidly alter temperature and hold samples at the desired
temperature for a set time. This automation is one of the important
advances that led to PCR becoming widely accessible to many scientists and
is covered in more detail in Chapter 3. Before thermal cyclers became avail-
able, PCR was performed by using three water baths set to temperatures of
typically 95°C, 55°C and 72°C, and reaction tubes in racks were moved
manually between the baths.
The other major technological advance that preceded the development
of thermal cyclers was the replacement of DNA polymerase I Klenow
fragment with thermostable DNA polymerases, such as Taq DNA poly-
merase, which are not inactivated at the high denaturation temperatures
used during PCR. The ability to carry out the reaction at high temperatures
enhances the specificity of the reaction (Chapter 4). At 37°C, where
Klenow works best, primers can bind to nontarget sequences with weak
sequence similarity, because mismatches between the two strands can be
tolerated. This leads to poor specificity of primer annealing and the amplifi-
cation of many nontarget products. The introduction of thermostable DNA
polymerases also reduced the cost of a reaction by reducing the amount of
polymerase required. With Klenow, at each denaturing step the enzyme
was also denatured and therefore a fresh aliquot had to be added at each
cycle. Thermostable polymerases retain their activity at the denaturation
temperatures and therefore only need to be added at the start of the
reaction.
1.4 PCR applications and gene cloning
PCR has revolutionized our approach to basic scientific and medical
research, to medical, forensic and environmental testing. It provides an
extremely flexible tool for the research scientist, and every molecular