The Polymerase Chain
Reaction (PCR)
By
Dr. NAGLAA FATHY
Lecturer of Biochemistry & Molecular Biology
Faculty of Medicine
Benha University
E-mail : naglaa_fathy722000@yahoo.com
Nagla.alhusseini@fmed.bu.edu.eg
What is the Polymerase
Chain Reaction?
It’s a means of selectively amplifying a
particular segment of DNA.
 The segment may represent a small part
of a large and complex mixture of DNAs:

Invented by Kary Mullis
Mullis and Faloona, 1987. Specific
synthesis of DNA in vitro via a
polymerase-catalyzed chain reaction.
Nobel Prize 1993
Kary Mullis
Did He Really Invent PCR?
• The basic principle of
replicating a piece of DNA
using two primers had
already been described by
Gobind Khorana in 1971:–
Kleppe et al. (1971) J. Mol.
Biol. 56, 341-346.
• Progress was limited by primer
synthesis and polymerase
purification issues.
• Mullis properly exploited
amplification
PCR
Specifically targets and amplifies a
SINGLE sequence from within a complex
mixture of DNA.
 How
is this different from cloning?
Amplify DNA
PCR
In vitro amplification (in a test tube)
 Enzymatic: Taq polymerase
– Temperature-resistant DNA
polymerase ( Thermus aquaticus)
 Heat resistant
 Best for <2 kb target

Takes advantage of basic
requirements of replication




A DNA template
Nucleotides
Primers
polymerase
PCR is DNA replication in a test tube
PRIMERS
Primers: short ssDNA sequences
complementary to border of
sequence of interest
Primers
Must
have some information
about sequence flanking your
target
Primers provide specificity
Primers
 ends
pointing towards each other
 Complementary to opposite strands with
3’
 Should have similar melting temperatures
PCR
Region of interest:
between primers
2. Anneal
3. Extend
Taq polymerase: enzymatic extension
PCR
Repeated Cycles of
1.Denaturation
1. Denaturation
2. Annealing
3. Extention
2. Anneal
3. Extend
Melting temperature
TmoC :Temperature at which
half possible H bonds are
formed.
TmoC = 2(A+T) + 4(G+C)
5/ - AGACTCAGAGAGAACCC-3/
4Gs 5Cs 7As 1T
TmoC= (4x9) + (2x8) = 36+16 = 520C
Annealing T =Tm0C -5
Heat-stable polymerase is vital
to the ease of the process…
 Thermus
aquaticus
Thermus aquaticus from hot springs in
Yellowstone National Park, USA.
The Thermus
aquaticus DNA
polymerase
Taq
 Not permanently
destroyed at 94ºC
 Optimal
temperature is 72ºC
Problems with Taq
Taq DNA polymerase - thermostable
 Lack of 3′-5′ exonuclease – proofreading
►Error rate = 2 × 10-4 nucleotdes/cycle
 Newer polymerases have high fidelity
High fidelity polymerase - HiFi Taq
Termplates for PCR
 Small amount of template

In theory a single molecule
 Do not need to isolate sequence of interest
 DNA template need not be highly purified
 DNA is stable in absence of nucleases
Templates for PCR
􀂄 Dried blood
􀂄 Semen stains
􀂄 Vaginal swabs
􀂄 Single hair
􀂄 Finger nail scrapings
􀂄 Egyptian mummies
􀂄 Buccal Swab
􀂄 Tooth brushes
Basic reaction
►Thermocycling, PCR machine
􀂃 Previously – need to overlay oil
to prevent evaporation
􀂃 Automatically Change
temperature
􀂃 Temperature gradient
The Basics of PCR Cycling
• 30–35 cycles each
comprising:
– denaturation (95°C),
30 sec.
– annealing (55–60°C),
30 sec.
– extension (72°C),
time depends on
product size
How many copies?
• No target products are made until the third
cycle.
• The accumulation is not strictly a doubling
at each cycle in the early phase.
• At 30 cycles there are 1,073,741,764 target
copies (~1×109).
How many cycles?
• Increasing the cycle
number above ~35 has
little positive effect.
• The plateau occurs when:
– The reagents are depleted
– The products re-anneal
– The polymerase is
damaged
- Unwanted products
accumulate.
Basic reaction

Oligonucleotide
primers
 Design to flank the
desired sequence
 Steps include: (3040 steps)
􀂄 Denaturation at
94°C
􀂄 Primer annealing
at Tm-5°C
􀂄 Extension at 72°C
Shortcut to pcranimatie.lnk
rtPCR
Reverse Trascription PCR (RT-PCR)





Use mRNA as a template
Total cellular RNA - faster
Contamination of genomic
DNA – false result
Primer specific to exons
Treat sample with DNase
Can be quantitative
Multiplex PCR



Simultaneously modification of more than
one locus in the same reaction
Rapid and convenient – screening
Included different set of primers
Quantitative or Real Time PCR

Monitors the fluorescence emitted during the
reaction as an indicator of amplicon production
 during each PCR cycle.
􀂄 The parameter CT (threshold cycle) is defined
as
the cycle number at which the fluorescence
emission exceeds the fixed threshold
(background).
Quantitative or Real Time PCR
Three different
fluorescence systems:
►Hydrolysis probes
►Hybridizing probes
►DNA binding agents
SYBR-Green I
Molecular Beacons

Uses FRET

Fuorescence Resonance Energy Transfer
Uses two sequence specific
oligonucleotides labeled with fluorescent
dyes

In situ PCR
Applications of PCR
Mutation detection .
 Diagnosis or screening of acquired
diseases,: e.g. AIDS, HBV & HCV.
 Prenatal diagnosis
 DNA profiling in forensic science
 Quantitation of mRNA in cells or
tissues.

Problems with PCR
 Contamination


Theoretically one molecule can amplify
Takes one mismatch early on to amplify
the wrong fragment
Dr. Naglaa Fathy