The Polymerase Chain Reaction (PCR)

Maisy the research meerkat is pregnant, but who’s the daddy? 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. 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. DNA

What would she need to do to get a DNA sample? 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 temperature is reduced for this step. 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

+ Step 3: DNA is copied (temperature increased to 72°C) DNA polymerase Annealed primers +

Then start the three steps all over again, but this time the amplified DNA is also used as a template... …to give four...

With just one template you would get 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 Just to recap 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.

In the past this was done in tanks using agarose gel electrophoresis 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. This machine uses capillary gel electrophoresis which uses the same principles as gel electrophoresis carried out in electrophoresis tanks, apart from the tank is replaced by very thin capillaries which contain a polymer instead of agarose. The machine detects the fragments, using a laser, as they pass through the capillary. The fragments are labelled with fluorescent tags which are attached to the primers in the PCRreaction. This machine is made by Applied Biosystems, whose website gives further information about their models.

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. This is real data. The graph shows two main peaks from two different samples. The x axis gives the sizes of the fragments in base pairs. The Y axis is the intensity of the luminescence given off from the fluorescent tag which is attached to the PCR primers which enables the machine to detect the DNA fragments. 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 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 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.