The Polymerase
Chain Reaction
(PCR)
The problem!
Maisy the
research meerkat
is pregnant, but
who’s the daddy?
How can we find
out?
A real problem for a real
scientist
What did
she do?
Johanna Nielsen is a PhD student at
the University of Edinburgh and the
Institute of Zoology at the University
of Cambridge
She knew that DNA is the genetic material of living
things.
The DNA sequence of the foetus would therefore have
greater similarity with the father than with a male meerkat
who was not the father.
She had two candidate fathers and decided to compare
the DNA from the foetus with that of the two possible
fathers.
She used the DNA profiling (fingerprinting) technique,
which involves the use of the polymerase chain reaction
(PCR).
The DNA profiling
technique
compares the size
of DNA at a specific
site between
individuals.
DNA
Johanna knows that this
section of DNA can be of
different sizes between
meerkats. She wants to
compare the DNA at this
site between the three
samples.
Firstly, she needed to extract
the DNA from each potential
father and the foetus.
What would she need to do to
get a DNA sample?
By using PCR, Johanna can amplify
this section of DNA many times.
PCR is now fully automated so Johanna just
needs to put the DNA samples into separate
tubes in the PCR machine, which is called a
thermocycler, along with a PCR ‘master mix’,
which includes all of the ingredients the PCR
reaction needs.
PCR uses the same mechanism for copying
the DNA as our cells use for DNA
replication.
In pairs remind yourselves what happens
during DNA replication. What is the main
enzyme involved?
PCR uses the enzyme DNA polymerase to
replicate or ‘amplify’ the DNA strand.
The polymerase chain reaction
The DNA is heated to 94°C to
denature the double helix so
amplification can take place.
Step 1: DNA denatured
Primers are used to start the
replication process of just the
section needed.
Primers are short single strands of
DNA that bond or ‘anneal’ to the
sections of DNA on either side of
the section you want on the DNA.
The temperature is reduced for this
step.
Step 2: Primers anneal
DNA polymerase
Step 3:
DNA is
copied
Annealed primers
(temperature
increased to 72°C)
+
Then start the three steps all over again,
but this time the amplified DNA is also used
as a template...
…to give four...
This cycle is continued for 30–40 cycles.
If there were 35 cycles from one template strand
how many copies would you have after the final
cycle? (You might need your calculator!)
A lot!
With just one template you would get
34,359,738,368 copies after 35 cycles.
You will usually start off with more
than one DNA template from a
DNA extraction. Why?
Each cell has a copy of the DNA
and an extraction will be from
many cells.
A recap... make notes about the steps
30-40 cycles
Back to Maisy, who
is still pregnant!
After PCR what will be in the sample tubes?
Maisy’s baby
Malcolm
Martin
Millions of copies of the DNA section/fragment
selected by Johanna.
Remind yourself why Johanna wanted to
amplify this fragment.
Johanna needs to find out the size of the DNA fragments inside each
sample.
She uses a technique called DNA gel electrophoresis, which separates
DNA fragments based on their size.
In the past this was done in tanks using agarose gel electrophoresis
but laboratories now use an automated machine
that can process many samples quickly using
capillary gel electrophoresis.
The results are then processed by a computer, producing data that look
like the above. This example shows the results of two different PCR
samples. One of the DNA fragments is 121 base pairs in length, the other
159 base pairs.
If the PCR sample contains millions of DNA fragments, why would
one sample only produce one data peak?
Yes, that’s right, the PCR reaction will amplify the same fragment many
times. The technique, however, is not perfect and that is why sometimes you
get the extra little peaks that you can see in the data above.
Here is Johanna’s data for three different DNA
fragments from each of the meerkats
Martin!
Who is the daddy?
Malcolm
Martin
Answer yourself, then pair,
then share.
Draw an annotated diagram to
describe the steps of the PCR
reaction.
We can’t see it but on the skin of our
hands is DNAse. Why would it be
important for Johanna to wear
gloves during her experiments?
DNAse is an enzyme that breaks
down DNA. Johanna wants the
DNA intact so she can analyse it.
Johanna has to take care to make
sure the DNA samples are kept
separate so as not to contaminate one
with the DNA of another. Why is this so
important?
PCR can amplify one template DNA strand
millions of times. Even the smallest amount of
contamination from another DNA source
could create millions of copies of that DNA.
The result would then be from the wrong DNA!
This is the tube that Johanna is preparing for
the PCR reaction. She has already put the
DNA sample in there, but what else does she
need?
Think about the PCR reaction and the fact that
it will create millions of new strands of DNA.
To make copies of the DNA you need to add to the PCR
mix:
DNA polymerase – you probably got this one
nucleotides of each base type – hopefully you got that
buffer to keep the pH optimal – might not have got that
Mg+, which DNA polymerase needs to work (it’s a
cofactor) – you probably didn’t get that.
PCR uses a special DNA
polymerase – Taq polymerase
Why?
+
Where is Taq from?
+
What is one of the problems with
using it?
Use the internet to answer these questions. Paste Taq
polymerase into your search engine. You have 10 minutes to find
the answers!
Taq polymerase is stable at high temperatures and is
therefore perfect for PCR, where the first step of each
cycle requires temperatures of 94°C in order to denature
the DNA.
Taq polymerase comes from a thermophilic bacterium
Thermus aquaticus, which lives in hot springs or
hydrothermal vents, hence its high temperature tolerance.
The main problem with using Taq polymerase in PCR is
that it doesn't have a mechanism for proofreading the
DNA. This can lead to errors that are not corrected.
Remember that PCR produces millions of copies from just
one template. If a copy contains an error, for example a
change in one of the base pairs, then that error will be
copied millions of times, which is especially problematic if
it occurs in an early cycle.