POLYMERASE
CHAIN REACTION
• PCR is an in vitro technique for
generating large quantities of a
specified DNA.
• Karry Mullis, the inventor of
PCR in 1989, was awarded the
1993 Nobel Prize in Chemistry
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.
• Cell-free amplification for synthesizing multiple identical
copies (billions) of any DNA of interest.
• Basic tool for the molecular biologist.
• 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.
• Like Xerox machine for gene copying.
PCR Thermocycler
Thermocyclers
heated lids
adjustable ramping times
single/multiple blocks
gradient thermocycler blocks
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
PROCEDURE …..
PCR Reagents
• 1X Buffer
– 10mM Tris-HCl, 50mM KCl
• MgCl2
– 1mM - 4mM (1.5mM)
• dNTPs
– 200μM
• Primers
– 100nM-1μM, 200nm (or less) for real time analysis
• DNA polymerase
– Taq DNA polymerase is thermostable
– 1-4 Units (1 unit)
• DNA
– 10pg-1μg (20ng)
MgCl2 (mM)
1.5 2 3 4 5
Magnesium Chloride
(MgCl2 - usually 0.5-5.0mM)
Magnesium ions have a variety of effects
Mg2+ acts as cofactor for Taq polymerase
Required for Taq to function
Mg2+ binds DNA - affects primer/template interactions
Mg2+ influences the ability of Taq pol to interact with primer/template
sequences
PCR STEPS :
Steps:
[Link] (Separation):-
by heating at 94°C to 96 °C for 1 min..
2. Annealing (Priming):-
primers are annealed by cooling to temperature around Tm generally
between 40 to 60 °C most commonly 45°C for 1 minute .
3. Amplification (Polymerisation):-
DNA strands are synthesized by Taq polymerase
heat at 72°C for 1 min in presence of dNTPS.
Sources of DNA Polymerase:
In the original technique of PCR, Klenow fragments of
[Link] DNA polymerase was used.(The Klenow
fragment is a large protein fragment produced when
DNA polymerase I from E. coli is enzymatically cleaved by
the protease subtilisin)
This enzymes gets denatured at higher temp. therefore
fresh enzyme had to be added each cycle.
Therefore introduction of Taq DNA Polymerase (Lawyer
1989) from thermophilic bacterium, Thermus aquaticus.
Taq DNA Polymerase is heat resistant, hence it is not
necessary to freshly add this enzyme for each cycle to
PCR.
Polymerase Chain Reaction
TEMPLATES FOR PCR
• Dried blood and
bones
• Semen stains
• Vaginal swabs
• Fingernails
scarpings
• Buccal swab
• toothbrushes
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 primer . Primers added to •Primase
the reaction mix
Joining nicks . N/A as fragments are short
•Ligase
Denaturation
Raise temperature to 94oC
to separate the duplex form
of DNA into single strands
07/03/20
Design primers
• To perform PCR, a 10-20bp sequence on either
side of the sequence to be amplified must be
known because DNA pol requires a primer to
synthesize a new strand of DNA
07/03/20
Annealing
• Anneal primers at 50-65oC
07/03/20
Annealing
• Anneal primers at 50-65oC
Extension
• Extend primers: raise temp to 72oC, allowing Taq
pol to attach at each priming site and extend a
new DNA strand
Extension
• Extend primers: raise temp to 72oC, allowing Taq
pol to attach at each priming site and extend a
new DNA strand
07/03/20
Repeat
• Repeat the Denature, Anneal, Extension steps at their
respective temperatures…
07/03/20
Polymerase Chain Reaction
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
• 3’ end critical (new strand extends from here)
• 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
Variations of the PCR
• Colony PCR
• Nested PCR
• Multiplex PCR
• AFLP PCR
• Hot Start PCR
• In Situ PCR
• Inverse PCR
• Asymmetric PCR
• Long PCR
• Long Accurate PCR
• Reverse Transcriptase PCR
• Allele specific PCR
• Real time PCR
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.
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.
Applications of PCR
• Classification • Detection of
of organisms pathogens
• Genotyping • DNA
fingerprinting
• Molecular
• Drug discovery
archaeology
• Genetic matching
• Mutagenesis
• Genetic
• Mutation
engineering
detection
• Pre-natal
• Sequencing
diagnosis
• Cancer research
[Link] in clinical diagnosis:
specificity & sensitivity of PCR is highly useful for the
diagnosis of various diseases in humans.
Eg- Inherited disorders (genetic diseases), Viral diseases,
Bacterial diseases etc
i) Prenatal diagnosis of inherited diseases
ii) Diagnosis of retroviral infections
iii) Diagnosis of bacterial infections
iv) Diagnosis of cancers
v) Sex determination of embryos
2. PCR in DNA sequencing
3. PCR in forensic medicine
4. PCR in comparative studies of genomes.
Thank you
THANK YOU
