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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
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.
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
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!