Lecture 9:

Polymerase Chain Reaction

(PCR)

Overview
PCR : is a molecular biology technique to amplify a
short region of a DNA molecule by million fold or
more
PCR can be used to clone a given DNA sequence in
vitro without the use of living cells during the
cloning process
Developers: Kary Mullis, who received a Nobel
Prize in 1993 for this work, invented the method in
1983

The Nobel Prize in Chemistry 1993 was awarded "for contributions to the
developments of methods within DNA-based chemistry

The Nobel Prize in Chemistry 1993

Polymerase Chain Reaction
The PCR method a
copying machine for DNA
molecules
DNA molecules can be massproduced from incredibly small
amounts of material with PCR.
Kary Mullis' discovery allows
the chemist to mimic the cell's
own natural DNA replication
process in a test tube. It has
now become much easier to
characterise and compare the
genetic material from different
individuals and organisms.

Principles of PCR
PCR uses synthetic oligonucleotides
complementary to known sequences to prime
enzymatic amplification of the intervening
segment of DNA in the test tube
Carried out by the DNA polymerase I enzyme
(normally from Thermus aquaticus)
This organism lives in hot springs, and many of
its enzymes, including the Taq polymerase, are
thermostable

 A repetitive series of cycles involving template

denaturation, primers annealing, and the
extension of the annealed primers by DNA
polymerase results in the exponential
accumulation of a specific fragment
Primer extension products synthesized in one
cycle serve as a template in the next cycles, thus,
20 cycles of PCR yields about a million-fold (220)
amplification

The Procedure
Most PCR methods typically amplify DNA
fragments from few hundred bp up to ~10 kb
A basic PCR set up requires several components
and reagents in a reaction volume of 10200l in
small reaction tubes (0.20.5 ml volumes)
The reaction is set up in a thin walled PCR tube
permit favorable thermal conductivity to allow for
rapid thermal equilibration in a thermal cycler

The Components of a PCR Reaction

DNA template that contains the DNA region (target)
to be amplified. Template DNA containing target
sequences can be added to PCR in single- or doublestranded form. PCR requires only a single copy of a
target sequence as template
Two synthetic oligonucleotide primers that are
complimentary to regions on opposite strands that flank
the target DNA sequence
A thermostable DNA polymerase that can withstand
heating 95oC or higher and to catalyse template-dependent
synthesis of DNA

 The four deoxyribonucleotides (dNTPs), the building-blocks

from which the DNA polymerase synthesizes a new DNA strand.
Standard PCR contains equimolar amounts of dATP, dTTP,
dCTP and dGTP (e.g., 20-200 M each, depend on experiment).
High concentrations of dNTPs (>4 mM) are inhibitory, because
of sequestering of Mg2+ ion
Buffer solution, providing a suitable chemical environment
for optimum activity and stability of the DNA polymerase
Divalent cations, magnesium or manganese ions; generally
Mg2+ . All thermostable DNA polymerases require free divalent
cations for activity. Because dNTP binds Mg2+, the molar
concentration of Mg2+ must exceed the molar concentration of
dNTPs. Usually 1.5 2.5 mM is used.
Monovalent cation potassium ions. Standard PCR buffer
contains 50 mM KCl

PCR Cycle

Three-step Cycling Process

Initial denaturation
Denaturation
Annealing
Extension
Final extension
Hold

Denaturation (Td):
The first step: thermal denaturation of the DNA sample
at 94-96oC for 30 s-1 min
Double-stranded DNA template denature at a
temperature that is determined in part by their G + C
content
The higher the proportion of G + C, the higher the
temperature required to separate DNA strands
The longer the DNA molecules, the greater the time
required at the chosen denaturation temperature to
separate the two strands completely

 If the temperature for denaturation is too low or if

the time is too short, only AT-rich regions of the
template DNA will be denatured
When the temperature is reduced later in the PCR
cycles, the template DNA will re-anneal into a fully
native condition
At 94-95oC, Taq polymerase can only endure for
~30-40 cycles without sustaining excessive damage

Annealing (Ta) of primers to template DNA

If the annealing temperature (Ta) is too high, the
oligonucleotide primers anneal poorly to the template
and the yield of amplified DNA is very low
If Ta is too low, non-specific annealing of primers may
occur
Annealing is usually carried out 3-5oC lower than the
calculated melting temperature (Tm)
The length and nucleotides contents (especially G + C) of
the primers used in PCR determine the Ta

 The time of annealing process (~30-60s) also determine

by the length and nucleotides contents of the primers

Extension of Oligonucleotide Primers

Is carried out at 72-78oC
In the first two cycles, extension from one primer
proceeds beyond the sequence complementary to the
binding site of the other primer
From the third cycle onward, this segment of DNA is
amplified geometrically or exponentially
The polymerisation rate of Taq polymerase is ~2000
nucleotides/minutes at the optimal temperature (7278oC), but in experiment, extension is carried out for 1
minute for every 1000 bp of product

Final Extension and Hold

Final Extension and Hold

Usually after the last cycle of PCR, an extension
time of 5-10 min is carried out to allow the
completion of all amplified products
Lastly, the temperature is brought down to 4-15
o
C until the sample is taken out

Number of Cycles
The number of cycles required for amplification
depends on the number of copies of template DNA
present at the beginning of the reaction
The reaction proceeds until one of the components
becomes limiting
At least 25 cycles are required to achieve acceptable
levels of amplification of single-copy target sequences
Usually is between 25-40 cycles

PCR Cycle

Initial denaturation 95oC

2 min
Denaturation
95oC
30 s
Annealing
50-60oC 30s
Extension
72oC
1 min/kb
Final extension 72oC
5-10 min
Hold
4-15oC

25-40
cycles

The Thermal Cycler

 The thermal cycler heats and cools the reaction tubes to

achieve the temperatures required at each step of the
reaction
Many modern thermal cyclers make use of the Peltier
effect, which permits both heating and cooling of the block
holding the PCR tubes simply by reversing the electric
current
Most thermal cyclers have heated lids to prevent
condensation at the top of the reaction tube
Older thermo cyclers lacking a heated lid require a layer of
oil on top of the reaction mixture or a ball of wax inside the
tube

Summary
1.
2.
3.
4.

Principles
Procedure
7 Components of PCR
PCR Cycle

End of Lecture 9
Thank You