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