Polymerase Chain Reaction

(PCR) and Its Applications

Dr Saquiba Yesmine
 PCR
• Polymerase chain reaction, or PCR, is a
technique to make many copies of a specific
DNA region in vitro.
 What is PCR?
PCR is an exponentially progressing synthesis
of the defined target DNA sequences in vitro.

It was invented in 1983 by Dr. Kary Mullis, for

which he received the Nobel Prize in Chemistry
in 1993.
 What is PCR?

It is called “polymerase” because the

only enzyme used in this reaction is
DNA polymerase.
 What is PCR?

It is called “chain” because the products

of the first reaction become substrates of
the following one, and so on.
 PCR
• PCR method can generate of billions of copies of a particular
DNA fragment – called the sequence of interest/ DNA of
interest, or target DNA from a DNA extract -DNA template.
• If the sequence of interest is present in the DNA extract, it is
possible to selectively replicate it in very large numbers.
• DNA extracted from an organism or sample containing DNAs
of various origins is not directly analyzable. It contains many
mass of nucleotide sequences.
 If the sequence of interest is present in the DNA extract,
it is possible to selectively replicate it in very large
numbers

DNA extracted from an organism or sample containing DNAs

of various origins is not directly analyzable. It contains many
mass of nucleotide sequences
 PCR: The Reaction Components

1) Target DNA - contains the sequence to be amplified.

2) Pair of Primers - oligonucleotides that define the sequence

to be amplified.
3) dNTPs – deoxynucleotide triphosphates: DNA building
blocks.
dNTPs provide both the energy and the nucleotides needed for
DNA synthesis during the chain polymerization. They are
incorporated in the reaction medium in excess.
 dNTPs

The four dNTPS (dATP, dCTP, dGTP and dTTP) are added
by Taq polymerase to a template strand of DNA at the corresponding base
 PCR: The Reaction Components

4) Thermostable DNA Polymerase - enzyme that

catalyzes the reaction
5) Mg++ ions - cofactor of the enzyme

6) Buffer solution – maintains pH and ionic strength of

the reaction solution suitable for the activity of the
enzyme
• PCR relies on a thermostable DNA polymerase, Taq
polymerase, and requires DNA primers designed
specifically for the DNA region of interest.
• In PCR, the reaction is repeatedly cycled through a series
of temperature changes, which allow many copies of the
target region to be produced.
• PCR has many research and practical applications. It is
routinely used in DNA cloning, medical diagnostics, and
forensic analysis of DNA.
 Application of PCR?
• Polymerase chain reaction (PCR) is a common
laboratory technique used to make many copies of
a particular region of DNA.
• This DNA region can be anything the experimenter
is interested in. For example, it might be a gene
whose function a researcher wants to understand, or
a genetic marker used by forensic scientists to
match crime scene DNA with suspects.
 The goal of PCR
• Typically, the goal of PCR is to make enough of
the target DNA region that it can be analyzed or
used in some other way.
• For instance, DNA amplified by PCR may be sent
for sequencing, visualized by gel electrophoresis,
or cloned into a plasmid for further experiments.
 Taq polymerase
• Its DNA polymerase is very heat-stable and is
most active around 70°C (a temperature at which
a human or E. coli DNA polymerase would be
nonfunctional).
• This heat-stability makes Taq polymerase ideal
for PCR.
 PCR primers
• Like other DNA polymerases, Taq polymerase
can only make DNA if it is given a primer.
• In a PCR reaction, the experimenter determines
the region of DNA that will be copied, or
amplified, by the primers of choice.
 The steps of PCR
• The key ingredients of a PCR reaction are Taq
polymerase, primers, template DNA, and
nucleotides (DNA building blocks).
• The ingredients are assembled in a tube, along
with cofactors needed by the enzyme, and are put
through repeated cycles of heating and cooling
that allow DNA to be synthesized.
 Steps of PCR
• Denaturation (96°C): Heat used to separate DNA
strands. This provides single-stranded template for
the next step.
 Steps of PCR
• Annealing (55 - 65°C): The reaction is cooled down
so the primers can bind to their complementary
sequences on the single-stranded template DNA.
 Steps of PCR
• Extension (72°C): The reaction temperatures is
raised so Taq polymerase extends the primers,
synthesizing new strands of DNA.
 Steps of PCR
• Denaturation (96°C): Heat used to separate DNA
strands. This provides single-stranded template for the
next step.
• Annealing (55 - 65°C): The reaction is cooled down so
the primers can bind to their complementary sequences
on the single-stranded template DNA.
• Extension (72°C): The reaction temperatures is raised
so Taq polymerase extends the primers, synthesizing
new strands of DNA.
 PCR
• This cycle repeats 25- 35 times in a typical PCR
reaction, which generally takes 2 - 4 hours,
depending on the length of the DNA region being
copied.
 PCR

It is not just the original DNA that is used as a template each time. Instead, the new DNA that is
made in one round can serve as a template in the next round of DNA synthesis. There are many
copies of the primers and many molecules of Taq polymerase floating around in the reaction, so the
number of DNA molecules can roughly double in each round of cycling.
 The Reaction

PCR tube THERMOCYCLER

Denature (heat to 95oC)

Lower temperature to 56oC

Anneal with primers

Increase temperature to 72oC

DNA polymerase + dNTPs
Each round of replication leads to a
doubling of the amount of DNA in the
region between the primers.
However, the final PCR product does not
appear until the third round of
amplification.
At the end of third round, we get
correctly sized target sequence and four
templates are completely synthesized
for the first time.
 Primers

• Primers are single-stranded DNAs that serve as a

starting point for DNA replication
• The size of the primers is usually between 10
and 30 nucleotides in order to guarantee a
sufficiently specific hybridization on the
sequences of interest.
 Primers

• One of the primers is designed to recognize

complementarily a sequence located upstream of the
fragment 5′–3′ strand DNA of interest;
• The other to recognize, always by complementarity,
a sequence located upstream complementary strand
(3′–5′) of the same fragment DNA.
 Primers
• Primers should flank the sequence of interest
• Primers that match multiple sequences will give
multiple products
• A primer may form dimer with itself or with the
other primers
• Primers can have self-annealing regions within each
primers
• That can form hairpin preventing its annealing to
the target DNA.
 PCR product detection and
analysis
• The product of a PCR consists of one or
more DNA fragments (the sequence or
sequences of interest).
• The detection and analysis of the
products can be very
quickly carried out by agarose gel
electrophoresis.
• The DNA is revealed by ethidium
bromide staining
• Thus, the products are instantly visible
by ultraviolet transillumination (280–
Real-time PCR (Quantitative PCR in real
time )

Developed in the mid-1980s, quantitative

PCR can determine the level of specific DNA
or RNA in a biological sample.

The method is based on the detection of a

fluorescent signal that is produced in
proportion to the amplification of the PCR
product, cycle after cycle.
Real-time PCR (Quantitative PCR in real
time )

A nucleotide probe is synthesized so that it can

hybridize selectively to the DNA of interest
between the sequences where the primers
hybridize.

The probe is labeled on the 5′ end with a

fluorochrome signal (e.g., 6-carboxyfluorescein),
and on the 3′ end with a quencher (e.g., 6-carboxy-
tetramethyl rhodamine).

This probe must show temperature hybridization

Real-time PCR (Quantitative PCR in real
time )

The main advantage of real-time PCR over PCR is

that real-time PCR helps to determine the initial
number of copies of template DNA (the
amplification target sequence) with accuracy and
high sensitivity over a wide dynamic range.

Real-time PCR results can either be:

-qualitative (the presence or absence of a
sequence) or
-quantitative (copy number).
Real-time PCR (Quantitative PCR in real
time )

Quantitative real-time PCR is thus also known as

qPCR analysis. In contrast, PCR is at best
semiquantitative.

Real-time qPCR data can be evaluated without gel

electrophoresis, resulting in reduced bench time
and increased throughput.

Finally, because real-time qPCR reactions are run

and data are evaluated in a unified, closed-tube
qPCR system, opportunities for contamination are
reduced.
Nonspecific detection
Nonspecific detection
Real-Time Vs Traditional PCR
 Real-Time Vs Traditional PCR
Real-Time chemistries allow for the detection of
PCR amplification during the early phases of the
reaction.

Measuring the kinetics of the reaction in the early

phases of PCR provides a distinct advantage over
traditional PCR detection.

Traditional methods use Agarose gels for

detection of PCR amplification at the final phase
or end-point of the PCR reaction.
 Applications of PCR
• Classification of • Detection of
organisms pathogens
• Genotyping • DNA
• Molecular fingerprinting
archaeology • Drug discovery
• Mutagenesis • Genetic matching
• Mutation detection • Genetic
• Sequencing engineering
• Cancer research • Pre-natal
diagnosis
 Application of PCR
•Acellular cloning: One of the most remarkable
applications of PCR. It makes it possible to isolate, to
purify a gene without resorting to traditional methods of
molecular cloning which consist in inserting a DNA
library in a plasmid vector which is then used to transform
a bacterial strain whose clones after selection are
screened. The realization is much faster and much less
random using PCR. Acellular cloning is used when using
PCR because it is useless to use a cellular system
(bacteria, yeast, and animal or plant cell) to amplify the
clone.
 In diagnosis
Detection of genetic diseases
Detection of infectious diseases

PCR applied to identification

PCR is remarkably effective at identifying species,
varieties, or individuals by genetic fingerprinting.
This application is based on the knowledge
acquired on genome structure. It is simply to
amplify nucleotide sequences that are specific to
species, variety, or individual. I
Using gel electrophoresis to visualize the results of
PCR
The results of a PCR reaction are usually visualized
using gel electrophoresis.
Gel electrophoresis is a technique in which fragments
of DNA are pulled through a gel matrix by an electric
current, and it separates DNA fragments according to
size.
A standard, or DNA ladder, is typically included so
that the size of the fragments in the PCR sample can be
determined.
 Applications of PCR

PCR produces enough DNA copies to be analyzed using

other techniques.
For instance, the DNA may be visualized by gel
electrophoresis, sent for sequencing, or digested with
restriction enzymes and cloned into a plasmid
 Using gel electrophoresis to visualize the results of PCR

• DNA fragments of the same

length form a "band" on the gel,
which can be seen by eye if the
gel is stained with a DNA-binding
dye.
• For example, a PCR reaction
producing a 400 base pair (bp)
fragment would look like this on a
gel:
 Applications of PCR
The basic PCR procedure achieves two very useful objectives:
1.it can increase the amount of sample DNA considerably in a
relatively short period of time; and,
2.perhaps more importantly, it can be used to isolate a specific
region or sequence of DNA from a large amount of surrounding
or contaminating DNA.
This means, for example, that from a very small tissue sample –
perhaps containing only a few cells or even a single cell –
theoretically possible to amplify and isolate a specific gene or
DNA sequence for analysis.
 Advantages of PCR:Specificity
PCR:
1. The PCR is highly specific and can identify accurately even a
single base pair mutation in a DNA sequence containing
thousands of base pairs. The degree of specificity does,
however, depend upon the choice of
2. primers used in the reaction, and these need to be exact or
nearly exact matches for the desired DNA sequence to be
amplified.
3. primers that are 18 or more nucleotides in length should be
unique within a complex eukaryotic genome,
4. whereas primers as short as 10 nucleotides are likely to be
unique for any purified cloned DNA fragment.
 Advantages of PCR: Rapidity
- Results are possible within hours with this
method (e.g. a 30-cycle amplification of a 200-bp
DNA sequence can be completed within 3 hours).
- whereas conventional techniques require many
days or even weeks.
- Also, large numbers of samples can be analysed
at the same time
 Advantages of PCR: Versatility
-although the 3énd of a primer needs to be very specific, there is a
certain amount of adaptability in the rest of the primer.
- by reducing the reaction stringency, a primer can be used to
amplify slightly differing sequences.
- the 5énd of the primer can actually be totally different
from the sample sequence and so it is possible to add on
sequences to the ends of a PCR product.
- This method is used for introducing mutations into genes
 Advantages of PCR: Ease of use

-Once the reactants have been added together, all

subsequent steps are fully automated.
- The reaction tube is placed into a thermocycler, which
is programmed to complete the required number of
cycles.
 Advantages of PCR: Quantity
-It is possible to analyse a large number of samples at
one time or
-- to analyse a single sample for the presence of multiple
genes or
- mutations simply by adding multiple primer pairs to the
same sample
 Disadvantages of PCR: Identity of the DNA sequence
-The DNA sequence of interest must be partly known or
at least be relatively well predicted. However, since it
generally is possible to predict
-the sequence of a gene of interest, or at least a part of it,
from homologous genes with similar activities.
• Size of the sequence
-There is a limit to the length of sequence. Generally, this
is in the region of about 0.1–3kb,
-although larger fragments (up to 35kb) can be obtained
under ideal conditions. Taq DNA polymerase is not
 Disadvantages of PCR: Number of cycles
-If too many cycles are carried out when analysing very
small quantities of DNA, such as from a single cell,
- primers and dNTP levels may become limiting or the
Taq DNA polymerase may lose its activity

Contaminating DNA
-Contamination is a serious concern in PCR reactions and
must be avoided at all costs
 Applications of PCR
It is important to stress that no one individual protocol will be optimal for
all PCR reactions, nor will any single, simple set of variables to be
optimised necessarily produce a functionary protocol for a specific case.
However, the basic PCR procedure achieves two very useful objectives:
firstly, it can increase the amount of sample DNA considerably in a rela-
tively short period of time; and,
secondly, and perhaps more importantly, it can be used to isolate a
specific region or sequence of DNA from a large amount of surrounding
or contaminating DNA. This means, for example, that from a very small
tissue sample – perhaps containing only
 Applications of PCR
• Classification of • Detection of
organisms pathogens
• Genotyping • DNA
• Molecular fingerprinting
archaeology • Drug discovery
• Mutagenesis • Genetic matching
• Mutation detection • Genetic
• Sequencing engineering
• Cancer research • Pre-natal
diagnosis
 Applications of PCR
Basic Research Applied Research
• Mutation screening • Genetic matching
• Drug discovery • Detection of pathogens
• Classification of organisms • Pre-natal diagnosis
• Genotyping • DNA fingerprinting
• Molecular Archaeology • Gene therapy
• Molecular Epidemiology
• Molecular Ecology
• Bioinformatics
• Genomic cloning
• Site-directed mutagenesis
• Gene expression studies
 Applications of PCR

Molecular Identification Sequencing Genetic Engineering

• Molecular Archaeology • Bioinformatics • Site-directed mutagenesis
• Molecular Epidemiology • Genomic cloning • Gene expression studies
• Molecular Ecology • Human Genome Project
• DNA fingerprinting
• Classification of organisms
• Genotyping
• Pre-natal diagnosis
• Mutation screening
• Drug discovery
• Genetic matching
• Detection of pathogens
MOLECULAR IDENTIFICATION:
 Conclusion
The speed and ease of use, sensitivity,
sensitivity specificity and
robustness of PCR has revolutionised molecular biology
and made PCR the most widely used and powerful
technique with great spectrum of research and
diagnostic applications.