Topics
• What is the Polymerase Chain Reaction?
The Polymerase Chain
Reaction (PCR)
Raymond Dalgleish
Department of Genetics
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:
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
(visible by gel electrophoresis) 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.
• History and (pre-history) of PCR
• How PCR works
• Optimising PCR
• Fidelity, errors and cloning
• PCR primer design
• Applications of PCR
A Molecular Photocopier
• A photocopier capable of duplicating a part
of a sentence:
• “The next day was quite a different day. Instead of being
hot and sunny, it was cool and misty. Pooh didn’t mind for
himself, but when he thought of all the honey the bees
wouldn’t be making, a cold misty day always made him
feel sorry for them.” A.A. Milne, 1928.
• The words in blue must be unique for the
copier to locate the correct piece of text.
Gene Analysis Prior to PCR?
• Southern blotting (1975) permitted
rudimentary mapping of genes in unrelated
individuals (RFLPs, insertions & deletions).
• DNA sequencing (1978) required genes to
first be cloned into plasmid or λ vectors.
• Gene library construction and screening
could take many months and libraries had to
be made for each individual analysed.
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 The Invention of PCR Did He Really Invent PCR?
• Invented by Kary • The basic principle of replicating a piece of
Mullis in 1983. DNA using two primers had already been
• First published described by Gobind Khorana in 1971:
account appeared in – Kleppe et al. (1971) J. Mol. Biol. 56, 341-346.
1985.
• Progress was limited by primer synthesis
• Awarded Nobel
and polymerase purification issues.
Prize for Chemistry
in 1993. • Mullis properly exploited amplification.

The Basics of PCR Cycling What’s in the Reaction?

• 30–35 cycles each • Template DNA
comprising:
• Reaction buffer (Tris, ammonium ions
– denaturation (95°C),
30 sec. (and/or potassium ions), magnesium ions,
– annealing (55–60°C), bovine serum albumin)
30 sec. • Nucleotides (dNTPs)
– extension (72°C),
time depends on • Primers
product size. • DNA polymerase (usually Taq)

PCR In Detail How many copies?

• Denature, anneal, extend and repeat the
cycle 30 to 35 times.
• “How does the polymerase know to stop
when it reaches the other primer?”
• Most textbooks to not fully explain PCR.
• PCR animation at Dolan DNA Learning
Center, CSHL, Cold Spring Harbor.
• 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).
• There are also 60 other DNA copies.

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 How many cycles? So Then, it’s Easy?
• Increasing the cycle • Cycling performed with three water baths.
number above ~35 has
little positive effect.
• Thermal cyclers introduced in 1986.
• The plateau occurs when: • Early polymerases were not thermostable,
– The reagents are depleted so had to be replenished each cycle.
– The products re-anneal • The 37°C temperature caused non-specific
– The polymerase is priming, resulting in unwanted products.
damaged
• Unwanted products
• Taq (Thermus aquaticus) DNA polymerase
accumulate. first described in 1988.

Thermal Cyclers So, I Can Just Go Ahead?

•PCR cyclers available from many suppliers.
• Not so fast.
•Many block formats and multi-block systems.
• The PCR technique and the use of Taq
•Reactions in tubes or 96-well micro-titre plates.
DNA polymerase in PCR are both patented.
• Even academic and public organisations
must pay license fees — levy paid on
enzyme and thermal cycler purchases.
• Taq patent still being challenged (Promega).

Has It Worked? Optimising the PCR Reaction

• Check a sample by gel electrophoresis.
• Is the product the size that you expected?
• Is there more than one band?
• Is any band the correct size?
• May need to optimize the reaction
conditions.
• Annealing temperature of the primers.
• The concentration of Mg2+ in the reaction.
• The extension time.
• (The denaturing and annealing times.)
• (The extension temperature.)
• (The amount of template and polymerase
— “more is less”.)

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 Optimising the Annealing Optimising the Mg2+
Temperature Concentration
• Primers have a • The fidelity of the
calculated annealing PCR depends on
temperature [Mg2+].
(e.g. 54°C).
• Vary [Mg2+] in steps
• Temperature must be
of 0.5 mM.
confirmed practically.
• Temperature steps of • Sometimes a
2°C above and below. compromise between
• Use gradient cycler. yield and specificity.

Fidelity of the Reaction Do Errors Matter?

• 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.
• 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? Can I PCR Amplify RNA?

• Amplification products are typically in the
size range 100-1500 bp.
• Longer targets are amplifiable — >25 kb.
• Requires modified reaction buffer, cocktails
of polymerases, and longer extension times.
• Limited by the integrity of the starting
target DNA — > 50 kb.
• Not directly — the DNA polymerase
requires a DNA template and will not copy
RNA.
• mRNA can first be copied into cDNA using
reverse transcriptase.
• cDNA is a template for PCR — it need not
be double-stranded.

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 Cloning PCR Products
• Products should be ligatable into blunt-
ended restriction enzyme site.
• Lower than expected efficiency.
• Products are not truly blunt-ended.
• Taq polymerase adds a single non-
templated base (usually A) to the 3´ end:
NNNNNNN…NNNNNNNA
ANNNNNNN…NNNNNNN
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 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.
TA Cloning of PCR Products
• Take advantage of the non-templated bases.
• Linearise vector at a blunt-ended site (e.g.
EcoRV).
• Incubate linear vector with Taq polymerase
and dTTP to add non-templated Ts.
• Ligate:
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.
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.
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Primers for a COL3A1 variant
• The human COL3A1 gene has a variant at
amino acid 531 of the triple helix.
• Ala or Thr encoded in exon 31 of the gene.
• AluI restriction enzyme site present in the
Ala allele but absent in the Thr allele.
• PCR amplify the region and genotype by
digestion of PCR products with AluI.
The COL3A1 Ala/Thr PCR
• The PCR primers amplify from the start of
exon 31 to just beyond exon 33 — 656 bp.
• Ala alleles are digested by AluI, producing
fragments of 82 & 574 bp.
Virtual PCR Results
• Virtual PCR searches
entire genome looking
for potential primer
sites within 10,000
bases of one another.
• If found, it performs a
virtual PCR reaction.
• Primers for Ala/Thr
polymorphism in
human COL3A1.
Running Primer3
• Paste the DNA sequence into Primer3 with
the “target” enclosed in square brackets.
• Select a mispriming library — only human
and rodent available at present.
• Select option for a 1-base 3´ “GC Clamp”.
• Select PCR product size range (>600 bp).
• Click the “Pick Primers” button.
• Marvel at the ease and simplicity.
Will Other Genes Amplify
Too?
• The primers have been designed on the
basis of the DNA sequence of a single gene.
• Might the primers also amplify other
segments whose sequence we have not
taken into account?
• Need to consider the sequence of the entire
genome to answer this.
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.
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 Multiplex PCR
• PCR reactions can be devised in which
several targets are amplified simultaneously
— often used in diagnostic applications.