PCR ---The Polymerase

Chain Reaction

Zhang-HaiFeng
Department of Biochemistry
The Invention of PCR
 Invented by Kary
Mullis in 1983.
 First published
account appeared in
1985.
 Awarded Nobel
Prize for Chemistry
in 1993.
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.
Topics
 What is the Polymerase Chain
Reaction?
 History and (pre-history) of PCR
 How PCR works
 Optimizing PCR
 Fidelity, errors and cloning
 PCR primer design
 Applications of PCR
 What is the
Polymerase
Chain Reaction?
 It’s a means of selectively amplifying a
particular segment of DNA in vitro.
 The segment may represent a small part of a
large and complex mixture of DNAs:
e.g. a specific exon of a human gene.
 It can be thought of as a molecular
photocopier.
How Powerful is PCR?
 PCR can amplify a usable amount of
DNA in ~2 hours.
 The template DNA need not be highly
purified — a boiled bacterial colony.
 The PCR product can be digested with
restriction enzymes, sequenced or
cloned.
 PCR can amplify a single DNA molecule,
e.g. from a single sperm.
 Components of PCR Reaction
System?
 Template DNA
 dNTPs: dATP, dGTP, dCTP, dTTP,
 DNA polymerase (usually Taq)
 Primers
 Reaction buffer and magnesium ions
 Water: ddH2O

PCR is DNA replication in a test

tube
Templates 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
 Single hair
 Fingernail scrapings
 Insects in Amber (Jurassic Park)
 Egyptian mummies
 Buccal Swab
 Toothbrushes
 DNA polymerase for PCR
Heat-stable
polymerase
is vital
to the ease
of the
process…
Thermus aquaticus:
 The Thermus
aquaticus DNA
polymerase

 Taq

 Not permanently
destroyed at 94ºC

 Optimal temperature
is 72ºC
Problems with Taq
 Does not have proof readng ability
 Error rate 1 in 2 X 104 bases
 Seems rare but can be recovered in
cloning a single molecule
 Newer polymerases have high fidelity
Process of PCR
Three steps:
 denaturation,

 primer annealing,

 extension
 Step I: DNA denaturing
The three steps of PCR are carried out in the same tube,
but at different temperatures.The first step of the process
separates the two DNA chains in the double helix. This
is done simply by heating the tube to 95 oC for 30 seconds.

double-strand DNA single-strand DNA

 Step II: Primer annealing
The primers cannot bind to the DNA strands at such a high
temperature, so the tube is cooled to 55 oC. At this temperature,
the primers bind or "anneal" to the appropriate location in the DNA
strands.This takes about 20 seconds.

single-stranded DNA + primer annealed DNA

The final step of the reaction is to make a complete copy of the

templates. Since the Taq polymerase works best at 75 oC, the
temperature of the tube is raised.
 Step III: Primer extension
The Taq polymerase begins adding nucleotides to the primer and
eventually makes a complementary copy of the section of the
template that lies between the primers. This completes one PCR
cycle.

primer-annealed DNA primer-extended DNA

with Taq polymerase and
dATP, dTTP, dGTP, dCTP
 Thermal cycling
At the end of a cycle, each piece of DNA in the tube has been
duplicated. Since the cycle can be repeated 30 or more times and
each newly synthesized DNA piece can act as a new template,
one can obtain 230 or 1 million copies of a single piece of DNA.

amplified region
Note that the region of the DNA between the two primers will
be amplified. The flanking sequences not. The entire process
takes about 2-3 hours.
PCR amplification

The figure on the left shows the series of steps

in a single cycle. The exponential growth of
the double helical segment between the two
primers is illustrated above.
Taq polymerase

Taq Polymerase In Complex With Tp7, An Inhibitory Fab

 PCR Cycles Review
 Denaturation : 94- 95℃, 30 sec

 Primer Annealing: 55- 65 ℃, 30 sec

 Extension of DNA: 72 ℃,
time depends on product size
 Number of Cycles: 20-35
 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.
So Then, it’s Easy?
 Cycling performed with three water baths.
 Thermal cyclers introduced in 1986.
 Early polymerases were not thermostable, so
had to be replenished each cycle.
 The 37°C temperature caused non-specific
priming, resulting in unwanted products.
 Taq (Thermus aquaticus) DNA polymerase first
described in 1988.
 Thermal Cyclers
•PCR cyclers available from many suppliers.
•Many block formats and multi-block systems.
•Reactions in tubes or 96-well micro-titre plates.
Has It Worked?
 Check a sample by gel
electrophoresis.
 Is the product the size that you
expected?
 Is there more than one band?
 May need to optimize the reaction
conditions.
Optimizing the PCR
Reaction
 Annealing temperature of the
primers.
 The concentration of Mg2+ in the
reaction.
 The extension time.
Optimizing the Annealing
Temperature
 Primers have a
calculated annealing
temperature
(e.g. 54°C).
 Temperature must be
confirmed practically.
 Temperature steps of
2°C above and below.
 Use gradient cycler.
Optimising the Mg2+
Concentration
 The fidelity of the
PCR depends on
[Mg2+ ].
 Vary [Mg2+ ] in steps
of 0.5 mM.
 Sometimes a
compromise
between yield and
specificity.
Fidelity of the Reaction
 Taq DNA polymerase lacks the 3´→5´
proof-reading activity commonly present
in other polymerases.
 Taq mis-incorporates 1 base in 104.
 A 400 bp target will contain an error in
33% of molecules after 20 cycles.
 Error distribution will be random.
Do Errors Matter?
 Yes, if you want to clone the amplified
DNA — an individual molecule may
harbour several mutations.
 No, if you want to sequence the
amplified DNA or cut it with restriction
enzymes.
 Use a proof-reading thermo-stable
enzyme rather than Taq.
How Big A Target?
 Amplification products are typically
in the size range 100-1500 bp.
 Longer targets are amplifiable —
>25 kb.
Designing PCR Primers
 Primers should be ~20 bases long.
 The G/C content should be 45–55%.
 The annealing temperatures should be
within 1°C of one another.
 The 3´-most base should be a G or C.
 The primers must not base pair with each
other or with themselves or form hairpins.
 Primers must avoid repetitive DNA regions.
Primers That Form
Hairpins
 A primer may be self-complementary
and be able to fold into a hairpin:
5´-GTTGACTTGATA
||||| T
3´-GAACTCT
 The 3´ end of the primer is base-
paired, preventing it annealing to the
target DNA.
Primers That Form Dimers
 A primer may form a dimer with
itself or with the other primer.
5´-ACCGGTAGCCACGAATTCGT-3´
||||||||||
3´-TGCTTAAGCACCGATGGCCA-5´
 Primer dimers can be an excellent,
but unwanted, substrate for the
Taq polymerase.
Help With Primer Design
 Researchers agreed early on that the
design of PCR primers was difficult and
unreliable.
 Computer programs devised to take all
of the design criteria into account.
 Primer3 program at the Whitehead
Institute is the most reliable and
versatile tool currently available.
Applications of PCR
 Mutation testing, e.g. cystic fibrosis.
 Diagnosis or screening of acquired diseases,
e.g. AIDS.
 Genetic profiling in forensic, legal and bio-
diversity applications.
 Site-directed mutagenesis of genes.
 Quantitation of mRNA in cells or tissues.
Applications of PCR
 Detection of mutations
 screen for inherited disorders
 Detection of HIV
 Not standard test given
 Detect tuberculosis without culturing
 Prenatal sex determination
�
Applications of PCR
 Preimplantation diagnosis of
genetic
diseases
 Forensics
 Paternity testing
Thank you very much!