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Polymerase chain reaction

(PCR)
Dr Abdulla Bashein
2007-2008
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Theory
• Purpose: To amplify a specific region
of DNA to a high copy number (1 copy
of a sequence is not enough for us to
study)
• Use a DNA polymerase from a
bacterium to replicate the region for us
• Use heat to denature the “template”
DNA to permit replication (whereas the
cell uses enzymes)
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Primers
• During normal DNA replication in the
cell, DNA polymerases need primers
(WHY?)
• We make short oligonucleotide primers
(18 - 30 bases) to prime DNA replication
in vitro
• By using a two specific primers, we can
delimit the region to be amplified by the
polymerase
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PCR: Product=Template=Exponential Production

DNA P
dNTPs

DENATURE AND COOL TO ANNEAL (< Tm):

CYCLE

THEN ELONGATE:

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PCR: Product=Template=Exponential Production

Final PCR Product

• Primers
– Mismatch: change sequence
– Degenerate: relax constraints
– Multifunctional: add sequences
• Polymerases
– Thermostability
– Proofreading
– “Hot Start”

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• “Chain Reaction” because when we put

the reaction mixture through successive
temperature cycles there is a snowball
effect of amplification of our region of
interest.
• This temperature cycling involves a
denaturation step at 94ºC to melt the
template DNA (this temperature also
permanently denatures normal DNA
polymerases)

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• Next is an annealing step with a much

lower temperature to allow the primers
to anneal (what kind of bonding?) to
their complementary target sequence in
the template DNA.
• Finally, an extension step allows the
DNA polymerase to extend from the 3’
end of the primer, replicating the target
region

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Typical Ingredients for a PCR

reaction
Template DNA
Primers
Taq DNA Polymerase
MgCl2
Buffer (Tris, KCl)
dNTPs

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The Typical Cycle

• Denaturation: 94ºC
• Annealing: 55ºC
• Extension: 72ºC
• You can program a thermal cycler
machine to take your PCR reaction
tubes through successive cycles of
these temperatures, and just walk
away….

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Denaturation
• 94ºC is standard
• For 30 - 60 seconds
• The high heat stops enzymatic reactions
and melts DNA
• A longer (120 seconds +) initial
Denaturation step is common
• Taq does lose some activity over many
cycles

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Annealing
• A random process of Brownian motion!
• Hydrogen bonds are constantly being
formed and broken
• “Correct” annealing depends on:
– Concentration of primers (want huge
excess)
– Availability of annealing sites
– Presence of competing, non-ideal
annealing sites
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• Typical annealing temperature is

between 50 and 55ºC, for 15 -60
seconds
• But the optimal temperature depends on
the primer sequence and length!
• The TM of a primer is defined as: the
temperature at which, at an ideal
binding site, half of the possible h-bonds
are present
• Longer primers and/or higher G/C
content mean a higher TM - why?
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TM of a “perfect” primer =
4(#Gs + #Cs) + 2(#As +#Ts)

• Above the TM few primers are bound

• Below the TM most of the ideal sites

have primer bound, but so do many
non-ideal sites

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• Annealing of the primer at non-ideal

sites will lead to PCR artifacts:
amplified regions that are NOT your
target DNA

• Increasing the stringency of the PCR

reaction (= increasing the annealing
temperature) will usually eliminate
artifacts.

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Extension
• 72ºC for 30 - 90+ seconds
• Why don’t the primers just fall off at this
higher temperature before extension
begins?? (trick question)
• Taq can synthesize thousands of base
pairs per minute under ideal conditions,
so optimal extension time depends on
the length of your target sequence…

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• If your target sequence is:

500bp then extend for 30 seconds
500 - 1500bp then extend 60
seconds
>1500bp then extend 90 seconds
• Usually, it’s not possible to amplify
targets greater than around 2000 bp
using standard conditions and Taq (but
there are new protocols and
enzymes….)
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PCR experimental design

• Always include control tubes!

• What would be in the negative control?

• What would be in the positive control?

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So the thermal cycler is done,

now what? How do you know
what’s in there and if the PCR
worked?
• Run a few microliters of your reaction tube
contents on a minigel…

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• Band(s) mean you amplified something

• Your prior knowledge about the size of
your target is your best clue to whether
you are seeing artifacts or not.
• The next step is sequencing and (and/or
a Southern blot) to assess the validity of
your amplification

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7.3 DNA sequencing: the Maxam-Gilbert

method

Figure 7-27
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7.3 DNA sequencing: the Sanger

method

Four separate polymerization

reactions are performed

Figure 7-29a
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7.3 DNA sequencing: the Sanger

(dideoxy) method

Figure 7-29b,c Dr Abdulla Bashein 24