Polymerase Chain

Reaction (PCR)
Evros Polydorou
April 2018
What is PCR?
 Polymerase chain reaction (PCR) is a technique that is
used to make millions of copies of a particular section of
DNA or gene.

 The method was developed in the 1980s by the

American biochemist Kary Mullis, who got a nobel prize
for his work.

 PCR is exponential = 2ⁿ (n is the number of every cycle)

 How does PCR work?
 First of all there are five factors that are required in order
to set up a PCR:

1. DNA sequence of a specific region that has to be known.

2. DNA Primers: a short sequence of nucleotides that provides a
starting point for DNA synthesis by recognizing the base sequence
of the specific DNA region that will be amplified and binds to it via
complementary base pairing.
3. DNA nucleotide bases which are needed to create the new
strand of DNA. (dNTPs)
4. Taq polymerase enzyme to add in the new DNA bases.
5. Buffer to ensure the right conditions for the reaction. (DNA thermal
cycler to stabilize the temperature by heating and cooling)
PCR components
 Steps in PCR

1. Denaturation
2. Annealing of 3. Extension of
of DNA
primers DNA Molecules
template
 STEP 1:
This step consists of
heating the reaction to
a temperature of 94-96
°C, in order to make
the double-stranded
template of DNA it into
two single strands by
separating.
 STEP 2:
The reaction
temperature is
lowered to 50–65 °C
allowing annealing of
the primers to the
single-stranded DNA.
 STEP 3:
At this step Taq DNA
polymerase is added,
these primers are
extended at a
temperature of 72 °C
in 5'  3' direction,
resulting in a
quantitative doubling
of the DNA region(2ⁿ)
 Example
If 1000 DNA strands are going through
PCR, how much copies will we get after 20
cycles?
*Αssuming there is no primer dimer and surplus
concentration of them to take part in the
reaction (because they can alter our
calculations).
PCR is exponential
1. Each DNA multiplies in exponents of 2.
2. So for 20 cycles each DNA produces 220
copies.
3. Thus for 1000 DNA templates, we will have;
1000 x 220= 1000 x 1048576 = 10.5 x 108 copies
at the end of 20 cycles.
Uses of PCR
Creation of a genetic fingerprint: From a
Cloning experiments: sample of blood or semen, or from a hair
Firstly the specific region root. (also known as DNA profiling)
of DNA is cut off with its
specific primer pair by
the same restriction
enzyme. Then the DNA
with the primer binds to
a cloning vector, which
is a bacterial plasmid
and then it becomes a
recombinant DNA Genetic counselling: Screening the
plasmid which will parents for genetic disease before
undergo PCR again and deciding on having children.
create thousand of
copies.
 Summary
 PCR is a technique used to make a lot of copies of DNΑ.

 Usually PCR is used up to 30-40 cycles.

 Primers are usually RNA‘s, they always bind to 5‘-3‘ direction

and they are always a pair (forward and reverse).

 The change of temperatures is a must in PCR and that’s why it

has a machine called Thermal Cycler that changes the
temperature in each step.