Polymerase Chain Reaction

&
its applications

Dr. Ujjal Kumar Nath

Professor
Department of Genetics & Plant Breeding
Bangladesh Agricultural University
Mymensingh
• The Polymerase Chain Reaction
(PCR) was not a discovery, but
rather an invention
• A special DNA polymerase
(Taq) is used to make many
copies of a short length of DNA
(100-10,000 bp) defined by
primers
• Kary Mullis, the inventor of
PCR, was awarded the 1993
Nobel Prize in Chemistry
 Development….

• First reports using DNA polymerase

from Thermus aquaticus (1988)
• Taq-polymerase (Saiki et al, 1988) from
Yellow stone National Park hot springs
• Developed automatic “thermocycler” programmable
heat block
 Polymerase Chain Reaction (PCR)

• PCR is a technique which is used to amplify the number

of copies of a specific region of DNA, in order to
produce enough DNA to be adequately tested.

• The purpose of a PCR is to make a huge number of

copies of a gene. As a result, it now becomes possible to
analyze and characterize DNA fragments found in minute
quantities in places like a drop of blood at a crime scene
or a cell from an extinct dinosaur.
PCR Thermocycler
 What all PCR Can Do ?
• Starting with one original copy an almost infinite
number of copies can be made using PCR
• “Amplified” fragments of DNA can be sequenced,
cloned, probed or sized using electrophoresis
• Defective genes can be amplified to diagnose any
number of illnesses
• Genes from pathogens can be amplified to identify
them (i.e., HIV, Vibrio sp., Salmonella sp. etc.)
• Amplified fragments can act as genetic fingerprints
 PCR Reagents
• 1X Buffer
– 10 mM Tris-HCl, 50 mM KCl
• MgCl2
– 1 mM – 4 mM (1.5 mM)
• dNTPs
– 200 μM
• Primers
– 100 nM-1 μM, 200 nm (or less) for real time analysis
• DNA polymerase
– Taq DNA polymerase is thermostable
– 1-4 Units (1 unit)
• DNA
– 10 pg-1 μg (20 ng)
Polymerase Chain Reaction
 Initiation - Forming the
Replication Eye
Origin of Replication
5’ 3’
3’ 5’

3’ 5’
5’ 3’
3’ 5’
5’ 3’

3’ 5’
5’ 3’
3’ 5’
5’ 3’
 Extension - The Replication Fork
3’ 5’
5’ 3’
3’ 5’ 3’ 5’ Primase
Single strand
Laging Strand binding
5’ proteins
Okazaki 5’
fragment 3’
5’
RNA
Primers
DNA
Polymerase
5’
3’
Helicase

Leading Strand
5’
3’
 How are the functions of replication
achieved during PCR ???
Function PCR ENZYMES
 Melting DNA . Heat • Helicase
• SSB proteins
• Topoisomerase
 Polymerizing DNA . Taq Polymerase • DNA pol
 Providing . Primers added to
primer • Primase
the reaction mix

 Joining nicks . N/A as fragments • Ligase

are short
 PCR principle
Temperature
100
Melting
94 oC

50

0
T i m e
3’ 5’
5’ 3’
 PCR principle

Temperature
100
Melting
94 oC

50

0
T i m e
3’ 5’

Heat

5’ 3’
 PCR principle

Temperature
100
Melting Melting
94 oC Extension 94 oC
Annealing
Primers 72 oC
50 50 oC

0
T i m e
3’ 5’
5’

5’
5’ 3’
 PCR principle
30x
Temperature
100
Melting Melting
94 oC Extension 94 oC
Annealing oC
Primers 72
50 50 oC

0
T i m e
3’ 5’

Heat
5’

5’

Heat
5’
5’ 3’
 PCR principle
30x

Temperature
100
Melting Melting
94 oC Extension 94 oC
Annealing
Primers 72 oC
50 50 oC

0
T i m e
3’ 5’
5’

5’
5’

5’
5’

5’
5’ 3’
 PCR principle
30x

Temperature
100
Melting Melting
94 oC Extension 94 oC
Annealing
Primers 72 oC
50 50 oC

0
3’
5’
5’ T i m e
5’
5’
5’ 3’

Heat
5’

5’

Heat
5’
 PCR principle
30x

Temperature
100
Melting Melting
94 oC Extension 94 oC
Annealing
Primers 72 oC
50 50 oC

0
3’
5’
5’ T i m e
5’
5’ 5’
5’ 3’

5’
5’

5’
5’

5’
5’
 PCR principle
30x

Temperature
100
Melting Melting
94 oC Extension 94 oC
Annealing
Primers 72 oC
50 50 oC

0
3’
5’
5’ T i m e
5’
5’ 5’
5’ 3’

5’
Fragments of 5’

defined length
5’
5’

5’
5’
PROCEDURE …..
More Cycles = More DNA
Size Number of cycles
Marker 0 10 15 20 25 30
 PCR Optimisation 1: Buffers
• Most buffers have only KCl (50mM) and Tris
(10mM)
– Concentrations of these can be altered
– KCl facilitates primer binding but
concentrations higher than 50mM inhibit Taq

• DMSO, BSA, gelatin, glycerol, Tween-20,

Nonidet P-40, Triton X-100 can be added to aid
in the PCR reaction
– Enhance specificity, but also can be inhibitory

• Pre-mixed buffers are available

 PCR Optimisation 2: MgCl2
• MgCl2: required for primer binding
– MgCl2 affects primer binding, Tm of template DNA,
product- and primer-template associations, product
specificity, enzyme activity and fidelity

– dNTPs, primers and template chelate and sequester the

Mg ion, therefore concentration should be higher than
dNTPs (as these are the most concentrated)

– Excess magnesium gives non-specific binding

– Too little magnesium gives reduced yield

 PCR Optimisation 3: Primer Design
• Specific to sequence of interest
– Length 18-30 nucleotides
• Annealing temperature 50oC-70oC
– Ideally 58oC-63oC
• GC content 40-60%
• 3’ end critical (new strand extends from here)
• GC clamp (G or C at 3’ terminus)
• Inner self complementarity:
– Hairpins <5, dimers <9
• 3’ complementarity:
– <3-4 bases similar to other primer regions
 PCR Optimisation 4: Cycling
Conditions
• Denaturation:
– Some Taq polymerases require initial denaturation (hot
start)
• Annealing temperature:
– ~ 5oC less than Tm of primers
– Tm = 4(G + C) + 2(A + T)oC (or use of primer software)
– Decrease in annealing temperature result in non-
specific binding
– Increase in annealing temperature result in reduced
yield
 PCR Optimisation 5: Cycle Number
• 25-40 cycles Theoretical yield = 2n

• Half-life of Taq is ie. cycle 1 = 2, cycle 2 = 4, cycle 3 = 8, etc

eg. if you start with 100 copies after 30 cycles you
30 minutes at will have 107, 374, 182, 400 copies
95oC
• Therefore if you
use more than 30
cycles at
denaturation
times of 1 minute,
the Taq will not
be very efficient
at this point
In summary

• Primer length should not exceed 30 mer.

• Tm, not more than 60 degree .
• GC Content should be in the range of 40-60 %
for optimum PCR efficiency.
• Primers should end (3′) in a G or C, or CG or
GC: this prevents “breathing” of ends and
increases efficiency of priming.
 Types of PCR
Long PCR: Used to amplify DNA over the entire length up to 25kb of genomic DNA
segments cloned.

Nested PCR: Involves two consecutive PCR reactions of 25 cycles. The first PCR uses
primers external to the sequence of interest. The second PCR uses the product of the
first PCR in conjunction with one or more nested primers to amplify the sequence
within the region flanked by the initial set of primers.

Inverse PCR: Used to amplify DNA of unknown sequence that is adjacent to known
DNA sequence.

Quantitative PCR: Product amplification w r t time, which is compared with a

standard DNA.

Hot start PCR: Used to optimize the yield of the desired amplified product in PCR
and simultaneously to suppress nonspecific amplification.

Touch Down PCR: Touchdown (TD) PCR offers a simple and rapid means to optimize
PCRs, increasing specificity, sensitivity and yield, without the need for lengthy
optimizations and/or the redesigning of primers.
 Colony PCR
Colony PCR- the screening of bacterial (E. Coli) or yeast clones for
correct ligation or plasmid products.

Pick a bacterial colony with an autoclaved toothpick, swirl it into 25 μl

of TE autoclaved dH2O in an microfuge tube.

Heat the mix in a boiling water bath (90-100 °C) for 2 minutes

Spin sample for 2 minutes high speed in centrifuge.

Transfer 20 μl of the supernatant into a new microfuge tube

Take 1-2 μl of the supernatant as template in a 25 μl PCR standard

PCR reaction.
 Hot Start PCR
• This is a technique that reduces non-specific amplification
during the initial set up stages of the PCR

• The technique may be performed manually by heating the

reaction components to the melting temperature (e.g., 95°C)
before adding the polymerase

• DNA Polymerase- Eubacterial type I DNA polymerase, Pfu

• These thermophilic DNA polymerases show a very small

polymerase activity at room temperature.
 Nested PCR
• Two pairs (instead of one pair) of PCR primers are used to
amplify a fragment.

• First pair -amplify a fragment similar to a standard PCR.

Second pair of primers-nested primers (as they lie / are
nested within the first fragment) bind inside the first PCR
product fragment to allow amplification of a second PCR
product which is shorter than the first one.

• Advantage- Very low probability of nonspecific amplification

 Multiplex PCR
• Multiplex PCR is a variant of PCR which enabling
simultaneous amplification of many targets of interest in
one reaction by using more than one pair of primers.
 Inverse PCR
• Inverse PCR (Ochman et al., 1988) uses standard PCR
(polymerase chain reaction)- primers oriented in the
reverse direction of the usual orientation.

• The template for the reverse primers is a restriction

fragment that has been selfligated

• Inverse PCR functions to clone sequences flanking a

known sequence. Flanking DNA sequences are digested
and then ligated to generate circular DNA.

Application
• Amplification and identification of flanking sequences such
as transposable elements, and the identification of genomic
inserts.
 Long PCR
• Extended or longer than standard PCR, meaning over 5
kilobases (frequently over 10 kb).

• Long PCR is useful only if it is accurate. Thus, special

mixtures of proficient polymerases along with accurate
polymerases such as Pfu are often mixed together.

• Application- to clone large genes

 Reverse Transcriptase PCR

• Based on the process of reverse transcription, which

reverse transcribes RNA into DNA and was initially isolated
from retroviruses.

• First step of RT-PCR - "first strand reaction“-Synthesis of

cDNA using oligo dT primers (37°C) 1 hr.

• “Second strand reaction“-Digestion of cDNA:RNA hybrid

(RNaseH)-Standard PCR with DNA oligo primers.

• Allows the detection of even rare or low copy mRNA

sequences by amplifying its complementary DNA.
 Touchdown PCR
It is a method for increasing specificity of PCR reactions.

Touchdown PCR uses a cycling program where the annealing

temperature is gradually reduced (e.g. 1-2°C /every second
cycle).

The initial annealing temperature should be several degrees

above the estimated Tm of the primers.

The annealing temperature is then gradually decreased until it

reaches the calculated annealing temperature of the primers
or some degrees below.

Amplification is then continued using this annealing

temperature.
 Why real time PCR ?
• QUANTITATION OF mRNA
– northern blotting
– ribonuclease protection assay
– in situ hybridization
– RT-PCR
• most sensitive
• can discriminate closely related mRNAs
• technically simple
• but difficult to get truly quantitative results using
conventional PCR
 Real-Time PCR

Real-time PCR monitors the fluorescence emitted

during the reaction as an indicator of amplicon
production at each PCR cycle (in real time) as
opposed to the endpoint detection
• Traditional PCR has advanced from detection at
the end-point of the reaction to detection while the
reaction is occurring (Real-Time).

• Real-time PCR uses a fluorescent reporter signal

to measure the amount of amplicon as it is
generated. This kinetic PCR allows for data
collection after each cycle of PCR instead of only
at the end of the 20 to 40 cycles.
Real-time PCR advantages

* amplification can be monitored real-time

* no post-PCR processing of products

(high throughput, low contamination risk)
* ultra-rapid cycling (30 minutes to 2 hours)
* wider dynamic range of up to 1010-fold
* requirement of 1000-fold less RNA than conventional
assays
(6 picogram = one diploid genome equivalent)
* detection is capable down to a two-fold change
* confirmation of specific amplification by melting curve
analysis
* most specific, sensitive and reproducible
* not much more expensive than conventional PCR
(except equipment cost)
Real-time PCR disadvantages

* Not ideal for multiplexing

* setting up requires high technical skill and support
* high equipment cost
* intra- and inter-assay variation
* RNA liability
* DNA contamination (in mRNA analysis)
 Applications of PCR
• Classification • Detection of
of organisms pathogens
• Genotyping • DNA
• Molecular fingerprinting
archaeology • Drug discovery
• Mutagenesis • Genetic
• Mutation matching
detection • Genetic
• Sequencing engineering
• Cancer research
 Electrophoresis and
Movement of Molecules

• Molecules can have distinct charges

– Positive or Negative
– Net charge will cause different movement through
gel +

• Molecules can have different shapes

– Linear
– globular
– Alpha helix
 Macromolecular charge

• Macromolecules have a
variable net charge that
depends on pH
• pH at which net charge is
zero = pI V=
• Electrical shielding of charge
occurs when counterions
are solvated
V=
 Electrophoresis
• Horizontal Agarose Gels
• Agarose forms a gel or molecular sieve
that supports the movement of small
materials in solution used for DNA
• Vertical Polyacrylamide Gels
• Made of Polyacrylamide
• Used for Protein molecular size, shape,
charge
• IEF electrophoresis
• Western Blot technique
 Horizontal Gels
• Gel Box set up frequently used in DNA
analysis
 Agarose gels
• Usually used in DNA analysis
• Made up of linear polysaccharide mol wt
of 12,000
• Basic repeating unit is agarobiose
• Gels are prepared at 1% to 3% providing
tunnels for molecules to move through
• DNA can be much larger then most
proteins
Agarose Gel with DNA Bands
markers

• DNA is negatively
charged
• Smaller sized DNA
moves faster than
Larger DNA
• Markers are used
to determine
relative sizes of
DNA pieces
 PAGE
• Native : Protein is prepared with little
disturbance to the cellular material
– Proteins are associated
– Movement of samples through the gel can be
inconsistent
• SDS : Sodium Dodecyl Sulfate Is a detergent
– Protein coated with a negative charge in
proportion to its molecular weight
– Denatures and unfolds protein
– Reducing agents (DTT)break amino acid cross-links
 Uses for PAGE
• Separates proteins from each other
– Proteins separated by size
– Isoelectric point
• Determines
– Molecular size of protein
– Quantifies the amount present
– Displays Impurities
– Used in western blot assays by antigen
interactions
 Determine Molecular Weight
1. Run standard molecular weight markers
on gel
2. Run unknown protein on the same gel
3. Create a graph of the mol wt versus
distance molecule has moved
4. Using the distance the unknown has
moved determine the molecular weight
from graph
Molecular Weight Markers

Migration of molecular weight

of standards are compared to
unknown samplewt std vs
unknown
Polyacrylamide Electrophoresis
 SDS Effect on Protein Movement
• Sodium Dodecyl Sulfate denatures protein and
covers it with negative charges : moves to + end
• Vertical gels are designed so the top of the gel
box is attached to the negative power outlet
• The bottom of the gel box is attached to the
positive power outlet
• Movement through the PAGE gel is
proportional to mass not to charge
 Movement of Proteins on an SDS
Gel Protein Migration

-
Stacking of
proteins at top of Highest
gel at start Molecular
Wt. protein
Distribution of
proteins in a
charged field

+
Low weight
molecular dye
 % Polyacrylamide in Gel
• Gels can be made at different concentrations of
polyacrylamide
• Example: gels made at 3%,6%,9% and 12% will
produce different openings through which the
molecule will migrate
• The larger the opening allows large molecules
to move through the gel
Vertical Polyacrylamide Gel
Electrophoresis
Equipment for Electrophoresis
Gel Electrophoresis Equipment
Mini-PROTEAN Tetra Cell
Closed Mini Gel holder
 Open Gel Holder:
Allows New Gel to be Inserted
 Gel Holders
Placed in Mini-Protean Tetra
Cell
 Procedure in Short

LoadGe Place Buffer

Equip
Thank you

THANK YOU