The polymerase chain reaction (or PCR) is a technique for the in vitro

amplification of a desired sequence of DNA. PCR allows the generation of a large

quantity of DNA product (up to several µg) from only a few starting copies. It
has been shown that PCR can be used to generate a detectable quantity of DNA
from only one starting target (or template) molecule.
PCR was developed in the mid-1980's, but has already found multiple
applications, such as:
• Rapid amplification of intact genes or gene fragments
• Generation of large amounts of DNA for sequencing
• Generation of probes specific for uncloned genes by selective
amplification of a specific segment of cDNA
• Analysis of mutations for medical applications
• Detection of minute amounts of DNA for forensic purposes
• Amplification of chromosomal regions adjacent to genes of known
sequence
PCR theory
The PCR reaction is a DNA synthesis reaction that depends on the extension of
primers annealed to opposite strands of a dsDNA template that has been
denatured (melted apart) at temperatures near boiling. By repeating the
melting, annealing and extension steps, several copies of the original template
DNA can be generated.
The amount of starting material (target) needed is very small
It is not necessary to isolate the desired sequence, because it will be defined by
the primers that are used in the reaction. The primers are oligonucleotides
complementary to different regions on the 2 strands of DNA template (flanking
the region to be amplified).
The primer acts as a starting point for DNA synthesis. The oligo is extended
from its 3' end by DNA polymerase.
Primer design: Oligonucleotide primers used for PCR are usually between 20-30
nucleotides and have ~50% G+C content (melting temperature (Tm) ~55°C to
65°C). In most situations the primer pair consists of two single oligonucleotides,
but degenerate primers can be used if you do not know the exact DNA sequence
i.e. only the protein sequence is known or if you are trying to detect other genes
in a multigene family - only the 3'end of each primer has to match the target
sequence exactly. They are usually designed in order to anneal to regions in the
DNA sequence whose distance is ~ 500 bp or less - because short target
sequences amplify more easily. If longer fragments are necessary, may need to
optimise conditions (amplifying sequences longer than 10 kb are possible). If the
PCR product is to be cloned, then restriction enzyme sites can be incorporated
within the 5' ends of the primers. Several primer design computer programmes
are available. The stages of a PCR reaction

PCR is a cycle of three steps:

DENATURATION - the strands of the DNA are melted apart by heating to

95°C

ANNEALING - the temperature is reduced to ~ 55°C to allow the primers to

anneal to the target DNA

POLYMERISATION - the temperature is changed to the optimum temperature

in order for the DNA polymerase to catalyse extension of the primers, i.e. to
copy the DNA between the primers.

The cycle is repeated over and over again - as many times as needed to produce
a detectable amount of product.
Theoretically, one can double the number of target DNA molecules for each
cycle performed (2n, where n= # cycles). In reality, various factors (e.g.
decrease in [nucleotides] and [primers], loss of enzyme activity) mean that there
is not a perfect doubling of DNA copy numbers with each cycle.
The first attempts used Klenow fragment of E. coli DNA polymerase I as the
enzyme. This enzyme catalyses addition of nucleotides to the 3' end of the
primer and has negligible 3' -to- 5' exonuclease activity. However, the Klenow
fragment was not very successful. This enzyme is inactivated by the high
temperature (95°C) needed to 'melt' the DNA strands apart and so fresh
aliquots had to be added manually for each cycle of the reaction - which is very
tedious. In addition, the lower temperatures required for Klenow fragment
(37°C) to synthesise the new DNA strand led to the possibility that the primers
could anneal non-specifically to the wrong sequences (mismatching primers).
Amplification can occur when mismatching primers are close enough together on
opposite strands of DNA - and an unwanted sequence is produced with ends that
precisely match the primers. If such an 'incorrect' fragment is synthesised in
the early cycles of a PCR, it will be efficiently amplified on subsequent cycles.
Discovery of a thermostable DNA polymerase
The breakthrough came with the discovery of the thermostable DNA
polymerase Taq polymerase, from the thermophilic bacterium, Thermus
aquaticus, which lives in hot springs. Like the Klenow fragment, Taq polymerase
does not have a 3' -to- 5' exonuclease activity. However, this enzyme can resist
the high temperatures required to melt the template DNA apart without
denaturation (loss of activity) and works best at high temperatures (72°C). This
led to improved specificity & sensitivity. Annealing of primers to sites other
than the target sequence is significantly reduced at the higher temperatures
used for Taq polymerase.
The fact that only one aliquot ot Taq polymerase needs to be added to the PCR
reaction means that all components can be added to the tube at the start of the
reaction - allowing the automation of the PCR. Heating blocks which can be
programmed to carry out the time and temperature cycles for a PCR have been
developed (thermal cycler machine).
Template DNA
The integrity is more important than the amount (in other words, if the DNA is
severely degraded, it is unlikely to have intact target molecules.) Linear DNA is
amplified more efficiently than circular, so plasmid DNA should be digested
beforehand.
Almost any starting material can be used, provided it has not been exposed to
nucleases
* Can use DNA released by the boiling of cells
* Can use DNA from sources which have been preserved or stored, even for
several years
o resin/paraffin embedded
o dried blood spots
o Several thousand year old Egyptian mummies
o 10+ thousand year old freeze dried mammoths (well, maybe we're
pushing it here)
RT-PCR
RT-PCR is a reverse transcriptase step combined with PCR amplification and
uses RNA as starting material.
Typical PCR reaction conditions
A typical PCR reaction contains 5 components
1. reaction buffer
2. oligonucleotides
3. template DNA
4. dNTPs
 5. enzyme
This consists of Tris buffered reaction mixture (pH 8.3) containing 50 mM KCl +
1.5 mM Mg2+ (salt helps primer annealing); between 10 and 50 pmols each
oligonucleotide primer (same concentration of each); ~ 1ng of target DNA is
sufficient (105-106 molecules); 200 mM each of the 4 dNTPs (dATP, dTTP, dCTP
and dGTP); 2 Units of enzyme (1 Unit of activity corresponds to 1 µmol min-1
reaction rate)
Small amounts of neutral detergent and protein (bovine serum albumin) are often
added to help stabilise the enzyme.
Typical cycling regime
Approximately 20-40 cycles of the 3 reaction steps are performed in a PCR
reaction. A typical reaction sequence would be:
Initial denaturation - 95°C for 2 mins
30 cycles of
95°C (30 seconds denaturation)
55°C (30 seconds primer annealing)
72°C (60 seconds primer extension,
for fragments up to 1 kb)
Final extension of all DNA ends - 72°C for 10 mins
Storage of DNA - 4°C
Adjust parameters to suit requirements - If only a small number of target DNA
molecules are present in the sample, then more cycles can be performed to
generate a detectable amount of product. If the target DNA to be amplified is
larger than 1 kb, then longer extension times can be used.
• Calculate time required for extension step as ~1 minute per 1 kb sequence
for Taq polymerase
• If primers have trouble annealing, can change the Mg2+ concentration or
the annealing temperature.
* Can adjust Mg2+ concentration between 1-4 mM
* Annealing temperature of the primer ~ its Tm, can drop the
temperature 1° or 2°C if necessary
Specificity can be improved further
'hot-start' method - All the reagents except one are heated to 72°C before
adding the final one, usually the enzyme is added afterwards
Helps eliminate possible extension from mis-annealed primers at lower
temperatures.
'Nested primers' - primers designed to anneal to regions within the target
sequence amplified by the first round of PCR. Ensures that only the desired
sequence is amplified.
Important points to consider to ensure the desired result
Is the primer annealing temperature sufficiently high?
 • If the primer annealing temperature is too low, non-specific annealing can
occur and non-specific products can be formed.
Is Taq polymerase a suitable enzyme?
• Thermostable enzymes which have proof-reading facility (3' -to- 5'
exonuclease activity) are now available.
Fidelity determines accuracy of PCR amplification
DNA replication is not a perfect process. On occasion DNA polymerases will add
an incorrect nucleotide to the growing DNA strand. The rate of misincorporation
in a naturally replicating DNA molecule is approximately 1 in 109 nucleotides, this
extraordinary accuracy achieved through the proofreading facility of the DNA
polymerase.
In vitroTaq polymerase has no 3' -to- 5' exonuclease activity. With
temperature and salt concentrations typical of a PCR, the misincorporation rate
of ~1 in 104 nucleotides found with using Taq polymerase can introduce a
mutation into the target sequence. The enzyme canât distinguish these, which
leads to the amplification of sequences which contain the 'mutation' alongside
those of the original sequence.
Result is mixture of amplified sequences
This is less of a problem for many applications. Molecules with the same
misincorporated nucleotide will form a very small portion of the total number of
molecules synthesised. Misincorporation is important if PCR fragments are to be
used for cloning. Since each clone is derived from a single amplified molecule, if
the molecule contains one or more misincorporated nucleotides, then all the
cloned DNA in that clone will carry the identical mutation. Problem can be
reduced by starting with a large, rather than a small number of template
molecules. Fewer amplification cycles are needed, resulting in less DNA
synthesis.
Contamination can be a major problem
Strictly to be avoided. Contamination occurs when the target DNA is
inadvertently introduced into the reaction. This isn't a problem if the only
reason for amplifying the DNA is to make more of it, but it is a significant
problem if you are using PCR for a diagnostic test. This is why most PCR
reactions need to be set up in clean areas with dedicated instruments (such as
pipettors), glassware and plastics. DNA is stable, and can be carried through on
autoclaved plastics and can even 'fall off your fingertips' - skin cells shed from
the researcher. The most common source is the products of previous
amplifications. These can be carried in aerosol, particularly from pipettors and
tips.
Applications of PCR
1) Cloning a gene encoding a known protein
Primers can be designed from sequence of amino acids or gene sequence.
Amplified product can be used as a probe to pull out the full length gene from a
cDNA or genomic library
2) Amplifying 'old DNA'
Amplifying DNA sequences from museum material or fossils - look at evolution of
gene sequences (Molecular evolution studies)
3) Amplifying cloned DNA from vectors
Convenient way of checking the inserts - amplified DNA can be analysed by
electrophoresis, Southern blotting
4) Creating mutations in cloned genes
5) Rapid amplification of cDNA ends - RACE
Most clones in cDNA libraries are not full length. RACE enables the 5' or 3' end
of a transcript to be cloned - alternative to rescreening libraries for overlapping
clones. Only one gene specific primer is needed.
6) Detecting bacterial or viral infection
More sensitive than conventional diagnostic techniques (culturing samples from
patients or using antibodies to detect the presence of microorganisms and
viruses)
Important for detecting relatively small numbers of organisms
* AIDS infection
* Tuberculosis (Mycobacterium tuberculosis)
7) Cancer
Detecting mutations that occur in cancer and monitoring cancer therapy.
Determining if a patient is free of malignant cells
8) Genetic diagnosis
a. Diagnosing inherited disorders
* Cystic fibrosis
* Muscular dystrophy
* Haemophilia A and B
* Sickle cell anaemia
b. Diagnosing cancer - certain cancers are caused by specific and
reproducible mutations: e,g. Retinoblastoma - childhood cancer of the eye. The
heritable form (germ line mutation of one of the two retinoblastoma allelles):
mutation is detected in all cells. Spontaneous form: only detected in tumour
tissue.
c. Blood group typing
d. Prenatal diagnosis - such as determining the sex of foetuses for those at
risk of X-linked disorders