THE POLYMERASE CHAIN REACTION

 It is abbreviated PCR

 It is an in-vitro technique used in amplifying segments of DNA

 An indispensable tool in molecular biology and biotechnology

 is a technique in molecular biology used to amplify (multiply) a single

copy or a few copies of a piece of DNA, generating thousands to
millions of copies of that particular DNA sequence

 PCR is also referred to as molecular photocopying

 PCR is based on using the ability of DNA polymerase to synthesize

new strand of DNA complementary to the offered template strand of
DNA.

 PCR is a revolutionary method developed by Kary Mullis in the 1980s

THE POLYMERASE CHAIN REACTION
Components of a Polymerase
chain reaction

•All four ncleotides called

dNTPs

•Forward and reverse primers

•Enzyme Taq polymerase

•Buffer solution

•Template DNA sample to be

amplified.
 THE POLYMERASE CHAIN REACTION
PRIMER DESIGN

Primers are short oligonucleotide sequences

They are complementary to regions flanking our DNA fragment we want

to amplify

There are two primers, the forward and the reverse primer that anneal
to the region upstream and down stream of the DNA segment to be
amplified.

Primers bind to the 3’end of the fragment to be amplified and the

binding is such that the free 3’OH group of each prime points to each
other

DNA synthesizing enzymes cannot add nucleotides to build the

complementary strand with out a primer attached.
 THE POLYMERASE CHAIN REACTION
The sequences of the primers are complementary to the sequence of
the flanking region of the DNA fragment we wish to amplify.

The specificity of the PCR lies in the design of the two oligonucleotide
primers

Bioinformatics play a key role in effectively designing a primer

Properties to consider when designing successful primers include

The length of the primers

The annealing and melting temperature of primers

The G-C content of the primer

The secondary structure of the primer

 THE POLYMERASE CHAIN REACTION
Primer length

Best primer length should be between 18 and 30

Considered the optimum range to get the best yield

If the primer is too short, more non-specific DNA amplifications will be
formed

If it is too long the process of hybridization will be too slow

Annealing and melting temperature

Annealing temperature is the temperature that allows the primer to

base pair with the DNA
 • THE POLYMERASE CHAIN REACTION
 The melting temperature is the temperature where half of the primer
will dissociate from the DNA

 Generally, the annealing temperature should be 5 degrees below the

melting temperature so that most of the primers will be bound to the
template

 Low annealing temperatures may lead to non-specific binding

 The forward and reverse primers should ideally have similar melting
temperatures although ideally a difference of 2 or 5 is still acceptable

 Online tools are available to determine the annealing temperature but

can be done manually by adding 4 degrees for (G or ) and 2 degrees
for (A or T)
 Consider GATGGCT, Tm=4+2+2+4+4+4+2= 22
 THE POLYMERASE CHAIN REACTION
Make up of nucleotides (G-C content)

oG-C nucleotides pairs with their three hydrogen bonding provide or

accounts for strong bonding between between primer and template.

oThe ideal nucleotide composition of the primer between is 40-60% G-C

content

oThe G-C content is the percentage of Gs and Cs in the primer e.g. if a

primer contains 20bp, and has 6Cs and 4Gs, the G=C content therefore
is given by G+C content = 6Gs+4Cs
o= 10/20*100 = 50%

oIts also god to include a GC clamp at th 3’end of the primer. This

means you should have to include 2-3 Gs and Cs to the 3’end of the
primer if possible
 THE POLYMERASE CHAIN REACTION
Avoid formation of secondary structural elements

Depending on the primer sequence, there is the possibility of forming hairpins

or primer dimerizing with itself or formation of dimers between the two primers

Online tools are available to indicate the possibility of hair pins, self dimers or
cross dimers

Beware of repeats in nucleotide sequences as too many repeats may cause

misparing and primer can anneal to unwanted locations

Design proper
Soft wares are available to aid in primer design. A good example of
a software is the NCBI website www.ncbi.nim.nih.gov/assembly

You have to obtain the nucleotide sequence of a gene and you can
querry the system to design a primer based on precautions studied
above
 THE POLYMERASE CHAIN REACTION
POLYMERASES

Thermo-stable high fidelity DNA polymerases

Enzyme responsible for assembly of nucleotides to

create a DNA molecule is called DNA polymerases

Different types of DNA polymerases with different

attributes and are therefore useful in different types of
PCR reactions
 THE POLYMERASE CHAIN REACTION
POLYMERASES
Taq Polymerase

Source: Thermus aquaticus, a bacterium found in hot

springs.

Use:

Taq polymerase is heat-stable and is commonly used in

standard PCR. It is active at high temperatures, making
it suitable for the denaturation step in PCR cycles.
 THE POLYMERASE CHAIN REACTION
POLYMERASES

Pfu Polymerase:

•Source: Pyrococcus furiosus, a hyperthermophilic

archaeon.
•Use:
•Pfu polymerase has proofreading activity, which means
it can correct errors made during DNA synthesis. It is
often used in applications where high fidelity is crucial,
such as DNA sequencing
 THE POLYMERASE CHAIN REACTION
POLYMERASES

Tgo Polymerase:

•Source: Thermus sp.

•Use:

Tgo polymerase is often used in applications where

reverse transcription (RNA to cDNA) is combined with
PCR. It has both DNA polymerase and reverse
transcriptase activity
 THE POLYMERASE CHAIN REACTION
POLYMERASES

Klenow Fragment:
•Source: Escherichia coli (E. coli) DNA polymerase I.

•Use:

The Klenow fragment lacks the 5' to 3' exonuclease activity of

the full-length E. coli DNA polymerase I.

It is often used in techniques like DNA labeling and site-

directed mutagenesis.
 THE POLYMERASE CHAIN REACTION
POLYMERASES
TaqMan Polymerase

•Source

Modified versions of Taq polymerase.

Use

•TaqMan polymerase is designed for use in real-time

quantitative PCR (qPCR) assays. It is used in
combination with TaqMan probes to detect and quantify
the amount of DNA amplified during PCR in real-time
 THE POLYMERASE CHAIN REACTION
POLYMERASES

Phusion Polymerase:

Source:
•Fusion of DNA polymerase from Thermus aquaticus (Taq)
and Pyrococcus furiosus (Pfu).

•Use:
Phusion polymerase is a high-fidelity enzyme with enhanced
processivity.

It is used in applications where both high fidelity and robust

amplification are desired.
 THE POLYMERASE CHAIN REACTION

 Characteristics that define a polymerase

 Fidelity and processivity

Fidelity refers to the accuracy of DNA polymerase

during replication of amplicones and high fidelity is
achieved by having a proofreading mechanism and
a low mis-incorporation rate
 THE POLYMERASE CHAIN REACTION
 Characteristics define a polymerase
 Fidelity and processivity

 Fidelity refers to the accuracy of DNA polymerase during

replication of amplicones and high fidelity is achieved by having a
proofreading mechanism and a low mis-incorporation rate

 Fidelity affected by the PCR buffer components and thermocycling

conditions
 THE POLYMERASE CHAIN REACTION

Processivity is the number of nucleotides a polymerase

can incorporate before dissociating. DNA polymerases
with high processivity can add several nucleotides per
second before dissociating.
Processivity is affected by buffer conditions similar to fidelity
 THE POLYMERASE CHAIN REACTION

•Bst DNA polymerase is suited for loop-mediated

isothermal amplification

•
•Bsu DNA polymerase which is suited for isothermal
strand displacement applications
 THE POLYMERASE CHAIN REACTION
The dNTPs

Four deoxynucleotide triphosphates are essential in a PCR

reaction.
 THE POLYMERASE CHAIN REACTIoN
The dNTPs are bought as separate tubes containing indivual nucleotides
or come as a master mix with both the enzymes and the buffer

PCR Buffer components

The PCR Buffer is supplied as a 10X concentrate and should be diluted

1:10 in the final reaction (e.g., use 5 µl in a 50-µl PCR reaction). Buffer
Composition
•(10X): 200 mM Tris-HCl (pH 8.4), 500 mM KCl.
•50 mM Magnesium Chloride
 THE POLYMERASE CHAIN REACTIoN
Composition of the PCR reaction
-Equimolar amounts of the dNTPs

-Forward and reverse primer to a

final concentration of 2µM

-Taq polymerase

-Buffer

-Template DNA

-Dnase free water

Mixture is pipetted into the PCR tube and closed. The tube is
inserted into the thermocycler and the reactions conditions set as
required
 THE POLYMERASE CHAIN Reaction
The PCR process (conventional PCR)

Divided into 4 stages namely

Denaturation, primer annealing, elongation and detection

Denaturation
Denaturation occurs at about 94 degree Celsius and can be
divided into two stages

Initial denaturation which last for 3 mins and subsequent

denaturation which will last for 30 seconds

Aim is to denature double stranded into single strands

 THE POLYMERASE CHAIN Reaction
Primer Annealing

The next step after denaturation is primer annealing

The annealling temperature should ideally be 5 degrees less than the

melting temperature as already seen

Aim of this stage is for primers to anneal upstream and downstream

flanking regions at the 3’ end.

The primers will have a free 3’ OH group onto which polymerase can
add nucleotides

Primer annealing is a crucial step in PCR and one of the key reasons
why the reaction fails at times

Primer annealing takes place at about 50 to 60 degree depending on

Tm of primers
THE POLYMERASE CHAIN Reaction
Extension or elongation

Polymerase binds to the 3’ end of the

primer and extends it by adding
nucleotides in the 5’-3’ direction

Extension temperature is a function

of the enzyme and for some it 68
degress and others 72 degrees

Once extension is over in say 1

minute, the temperature is again
raised to 94 degree centigrage for the
whole process to start all over

This process will repeat 30 to 35

times
THE POLYMERASE CHAIN Reaction

We can see that at

each cycle, the
number of strands of
DNA double.

If the reaction runs

for 30cycles, the
number of DNA
strands formed will
be given by 2n
 THE POLYMERASE CHAIN Reaction
Others variations of PCR Multiplex PCR differ from conventional
a) Multiplex PCR PCR in that its amplifies more that
one target DNA using more than one
target primer pair

It’s a variant of conventional PCR

which enable simultaneous
amplification of many targets of
interest in one reaction by using more
than one primer pair

In this case we increase the amount

of starting material like dNTPs as
many reactions will be running at
once

The target sequences must be very

dissimilar else the possibility of primer
cross reaction being high
 THE POLYMERASE CHAIN Reaction
Types of multiplex PCR

Single template PCR reaction

 Single template with multiple primers amplifying different regions

of template. Template could be genomic DNA

 Multiple template PCR reactions

Multiple templates and several primer pairs in the same reaction

tube
 THE POLYMERASE CHAIN Reaction
Internal Controls
Potential problems in a simple PCR include false negatives due to
reaction failure or false positives due to contamination. False negatives
are often revealed in multiplex assays because each amplicon provides
an internal control for the other amplified fragments.

2. Efficiency
The expense of reagents and preparation time is less in multiplex PCR
than in systems where several tubes of uniplex PCRs are used. A
multiplex reaction is ideal for conserving costly polymerase and
templates in short supply.

3. Indication of Template Quality

The quality of the template may be determined more effectively in
multiplex than in a simple PCR reaction
 THE POLYMERASE CHAIN Reaction
Nested PCR

Type of conventional PCR that uses two pairs of primer pair

The first primer pair amplify the region or segment of interest plus
flanking sequences

The amplicones of the first PCR are used as template for the next round
of amplification using the next set of primers

The essence of carrying out a nested PCR is to avoid non-specific

binding of the primer and amplification of sequences undesired
sequences

The schematic representation of a nested PCR is as shown in the next

slide
 THE POLYMERASE CHAIN Reaction

Conventional PCR
 THE POLYMERASE CHAIN Reaction
unlike like conventional PCR
we can observe two primer
pairs represented as green
and red

Green primer used for the

outer PCR and the red primer
used for the nested PCR

The unwanted product

produced during the outer PCR
run will not be amplified by
the nested PCR as it will have
no complementary sequence
for the red primer hence it wll
be eliminated
 THE POLYMERASE CHAIN Reaction

The advantages of nested PCR include

Increased specificity due to the two rounds of amplification,
reduced non-specific amplification,

Enhanced sensitivity, especially when the initial target

concentration is low.
 THE POLYMERASE CHAIN Reaction

Limitations of nested PCR

requires more time and resources compared to a

single-round PCR.

Increased risk of contamination with primary amplicones

it requires more time and

resources compared to a single-round PCR.

 THE POLYMERASE CHAIN Reaction
Real time PCR-

Is a modified form of conventional PCR where the amplification is combined

with spectrofluorometry

This technique combines amplification of the DNA sample as well as

simultaneously measuring the amount of DNA produced after each cycle of
amplification-combines amplification and detection

The real time thermocycler consists of two parts linked together. A cycling unit
and cycling unit and a spectrofluorometer.

The spectrofluorometer excites specific fluorophores inside the sample and

then the fluorophore emits light which can be detected

The amount of detected light produced by the fluorophore is directly

proportional to the concentration of DNA sample in the tube
 THE POLYMERASE CHAIN Reaction
How does real time PCR work

Components of real time PCR include: template DNA, dNTPs mix, primers, DNA
polymerase and additionally a detector

The detector is the fluorophore detected by the spectrofuorometer

There are two types of detectors used in real time PCR: non specific detection
and specific detection

Non specific detectors are fluorescent dyes such as cybergreen

Cyber green binds non specifically to any double stranded DNA and when bound
to dsDNA it becomes fluorescent, emitting light at 520nm which can be
detected by a detector

Cybergreen binds to the minor groove of the dsDNA helix

 THE POLYMERASE CHAIN Reaction
When cybergreen is bound
to dsDNA it becomes
fluorescent but the unbound
is not.

The amount of fluorescence

produced by cybregreen is
proportional to the
concentration of dsDNA
after the PCR run

Cybergreen is
Cheap
Easy to produce
Effective
Sentive
Easy to handle

But
Non specific hence binds to
any DNA whether target or
non-target
 Other variations of conventional PCR
 Specific detection: detective probes
(TaqMan)

 On the right is a representation of

the Taqman probe

 It consists of a DNA strand attached

at the 5’end by a fluorophore and to
the 3’ end is a quencher.

 The quencher inhibits or absorbs

any fluorescence produced by the
fluorophore in as much as they are
in close proximity

 The probe is designed such that its

sequence is complementary to a
region of the DNA upstream the
primer binding site
 Other variations of conventional PCR
 Once the double stranded DNA helix is separated into two strands, the
TaqMan probe binds to its complementary sequence and the primers also
bind to the 3’ end of the DNA
Elongation by a specific polymerase with
5’-3’ exonuclease activity leads to release
of the fluorophore

Released fluorophore which is no longer

at the proximity of the quencher starts
fluorescing which can be measured

The remaining probe is eroded till the

quencher is released and the double helix
synthesized.

Advantage: specificity, can perform multiplex

RT PCR using different colours of fluorophore
for different genes

Expensive, false results as quenche cannot

stop fluorophore
 Other variations of conventional PCR
Reverse transcription PCR

Reverse transcription polymerase chain reaction (RT-PCR) is a laboratory

technique combining reverse transcription of RNA into cDNA and amplification
of specific cDNA targets using polymerase chain reaction

Mature mRNA contains poly A tail and a 5’ cap

Steps to carry out RT-PCR include

Create a DNA primer complementary to the 3’end of the modified mRNA. Such a
primer will be have a polyT sequence since the 3’ end has a polyA tail

Add the basic components of the RT-PCR reaction: template RNA, buffers, enzyme
reverse transcriptase dNTPs , polyTprimer, random hexamer primers

Reverse transcription PCR can be carried out in one step or two steps.
Other variations of conventional PCR
Other variations of conventional PCR
 Other variations of conventional PCR
Loop mediated isothermal amplification (LAMP)

uses 4-6 primers recognizing 6-8 distinct regions of

target DNA.

A strand-displacing DNA polymerase initiates synthesis

and 2 of the primers form loop structures to facilitate
subsequent rounds of amplification.
 Other variations of conventional PCR
https://www.neb.com/en/nebinspired-blog/getting-started-with-loop-mediated-isothermal-am
plification
Differences between LAMP and PCR