Welcome to the University of Liverpool
This is the building where most of our scientific research takes place and
where we are going to learn about the polymerase chain reaction
The technique is used to make several million copies of a single piece of DNA
It was discovered by Kary Mullis and is now widely regarded as one
of the most important and influential techniques in modern biology
We are first to understand how technique works and then we're going to
see how it's applied here at the University of Liverpool
The polymerase chain reaction
To get started we need our DNA sample
and three other main ingredients:
DNA polymerase
primers
and nucleotides
Nucleotides are the structural units of DNA and there are 4 types: T, A, C and G.
Each nucleotides is made of a sugar-phosphate backbone and
one of the four nitrogenous bases
Nucleotides are complementary to each other
A with T, C with G
This complementary
base pairing is the basis of double-stranded DNA.
Within our DNA sample
we have a target sequence of DNA which we are amplifying using PCR
Primers are single-stranded nucleic acid with roughly 20 bases
which flank the target DNA sequence. Two different primers are
required, one for the start the sequence being amplified and one for the end.
One end of a DNA polynucleotide is called 3-prime, the other 5-prime
but DNA polymerase only attaches to
the 3-prime end. Once attached to the 3-prime end of the primer
the DNA polymerase can initiate DNA synthesis.
This is our final PCR mixture ready to begin amplification.
First mixture is heated to 93 degrees Celsius. This breaks hydrogen bonds
between nucleotides in the DNA sequence.
This results in single-stranded DNA. The PCR mixture is then cooled to
55 degrees Celsius. This allows the primers to anneal to the ends of the target
DNA sequence.
The PCR mixture is then heated to 72 degrees Celsius.
This is the optimum temperature for the DNA polymerase to synthesize new
DNA with the nucleotides in the PCR mixture.
DNA synthease can begin synthesising
new complementary DNA strands beginning
at the primers.
This continues along the single-stranded DNA
Following this first cycle of PCR there are now 2 copies of the target DNA sequence.
This is our new PCR mixture and process is repeated.
The mixture is heated again,
cooled again to allow primers to anneal
and then heated again for DNA synthase to synthesize complementary strands.
After each cycle
the amounts of product has doubled.
This results in exponential amplification.
The process is usually repeated around 35 times
which could result in 68,000,000 copies.
So now we understand PCR
we can speak with one of the researchers to see how he uses the
technique on a daily basis to carry out his research.
As we progress through the 21st century,
there's a requirement for fuel and energy sources that will ultimately
replace fossil fuels.
Previously first-generation biofuels were thought to be the option. However
it turns out that these contributed more to the production of greenhouse
gases than they actually reduced.
There's now a real emphasis on research involving the production of
second-generation biofuels.
The main theme of second-generation biofuels is taking a by-product of food
crops, crops which we already grown and breaking them down to produce ethanol.
In order to do this it's essential to be able to identify novel micro-organisms
producing cellulases, the enzymes which degrade these by-products of the crops
which we use for food.
As part of my research we take landfill leachate
place cotton into it and leave it to incubate for 3 months.
The bacteria present in the landfill leachate begin to degrade and break-down
the cellulose.
These organisms do it by producing cellulases
so in our research by taking this
cotton string, our source of cellulose and extracting DNA from it
we use PCR to identify
the bacteria by amplifying specific genes, and through the identification of
these bacteria
we may be able to exploit them for the production
of second-generation biofuels and eventually fuels of the future.
So to begin with, this is an
extract of DNA which has been extracted from a piece of string
incubated in landfill leachate. String is pure cellulose
On the surface it is broken down by cellulose degrading organisms.
Cellulose degrading organisms are present. We extract DNA. This sample will be
from cellulose degrading organisms.
So we are going to extract DNA that will be from cellulose degrading organisms.
We'll use this DNA
in a universal PCR
reaction
using universal
16S rRNA primers
and we'll use this DNA as templates
in the PCR reaction
we have in the reaction
DNA, we have a forward and a reverse primer
which are required for the amplification of 16S rRNA gene.
We also have
a BioMix Red reaction mix and within the reaction mix is everything which is
required for the PCR to be carried out efficiently. This contains buffers,
dNTPs,otherwise known as nucleotides, so you've got your A, T, G and C in here.
Also the polymerase
required for the extension
of the DNA template.
So the PCR reaction is set up and carried out
in PCR tubes
that are
specifically designed so they fit into the PCR machine and seal properly.
We set PCR reactions up in either 25 microlitre or 50 microlitre
volumes.
So,into each tube we'll put DNA template
and forward and reverse primers,
It is essential you work quickly and
keep all the volumes the same.
Now the reverse primer
and finally
the PCR reaction mix which contains all components which are required
to carry out
the reaction. Once all the tubes contain all components
required, seal the lids.
I'll just mix everything.
It is essential that the reactions are placed on ice until you're are ready to put it
in the PCR machine.
This is the sample that's been made in the lab and it contains
everything that is required for
the PCR reaction. We will put it in the PCR machine now.
PCR machine automatically heat up and cool down samples based on what you have instructed it to do
so it will initially heat the samples to 95 degrees and cause
DNA, the double-stranded DNA to denature. It will then cool down
to an optimal temperature that is different for each primer, but normally 55 degrees
and this temperature will then allow
the primer to anneal
to the single stranded DNA template.
The machine will then heat back up again which will
allow the polymerase, the enzyme
included in the mix, then binds to the primer.
This will then allow the polymerase to move along the
single-stranded DNA template and elongate
hence creating more DNA molecules
and thus DNA template in the sample
Once the pcr reaction has finished,
you remove tubes from machine
and we use
gel separation equipment known as gel electrophoresis
to run the sample on an agarose gel
and this will allow us to find out what organisms are actually present
in the original DNA sample.
Once the samples have been loaded
onto the gel in the tank, we can put the lid
back on. We then run a
current of electricity
through the tank that allows DNA to move through the gel.
Once the DNA has been separated on the gel, we then use ultraviolet light
so we can visualize the DNA.
The DNA from the different bacteria which are present
in the original sample from the cotton.
As you can see from this gel, five different
bacterial species are present in the original sample
which was used to carry out PCR.
So now we understand PCR and also seen it in action trying to find fuels of tomorrow.
So I hope you'll agree that PCR is one of the most important techniques in modern biology.
END