When collecting a sample of DNA there is often a very small amount to play with. This can be very problematic for many biochemists for two main reasons.

Firstly if you are carrying out an experiment on the DNA (perhaps sequencing it, or inserting a small sequence of DNA) and it goes wrong, which can often happen with these techniques, you are in trouble. All the DNA needed for your experiment has been used up in a failed attempt! Secondly, by extracting DNA direct from the sample, it will contain all the DNA of the organism. More often than not, specific sections of DNA are required. Having large lengths of DNA can therefore make this process cumbersome and clumsy, decreasing the efficiency and increasing the possibility of error.

How are these problems overcome?

Using the Polymerase Chain Reaction (PCR) of course!

This is a staple biochemical technique which is commonly performed on small amounts of DNA before any experimentation is undertaken. PCR is able to amplify an experimenters desired segment of DNA exponentially, thus overcoming the two problems outlined above.

figure 1. Requirements for PCR

So, how does PCR work?

PCR proceeds in a series of cycles, and each cycle has three steps.

Denaturation at 95°C = high temperatures separate the DNA strands
Annealing at 55° C = lower temperatures allow the primers to bind to the template DNA
Extension at 72°C = the DNA polymerase and nucleotides can bind to synthesise a new DNA strand.

Each cycle doubles the number of DNA strands. The process is repeated over many cycles, which causes an exponential increase in the number of DNA strands.

All this scientific jargon can become very confusing, so I am going to breakdown the roles of each of the requirements of PCR.

Primers = specific sequences of DNA around 20 nucleotdies long. They all the DNA polymerase (in this case Taq polymerase) to bind to DNA and create a new strand. Primers are required for the specificity of PCR because they will only bind to their specific complementary base sequence.

Taq polymerase = a type of enzyme known as DNA polymerase, which initially binds to the primer, then incorporates the correct nucleotides into the new DNA strand. It is called Taq polymerase because it is taken from an organism called Thermus aquaticus. It is a special form of DNA polymerase that remains active at the high temperatures during PCR (most other DNA polymerases would be denatured).

But how does PCR amplify a specific segment of DNA?

This all down to the primers.

DNA has a polarity, meaning it has a 5’ end and a 3’ end. DNA polymerase enzymes are only able to synthesise new DNA strands in one direction (5’ to 3’). Since DNA strands run in opposite directions the primers that attach either side of the target region result in DNA polymerase working in opposite directions. As shown in figure 2 below.

Amplification of template DNA.png
figure 2. Amplification of template DNA

This means that by repeating the PCR reaction of many cycles, only the region between the red primers will be exponentially amplified (the darker blue lines in figure 2). This is demonstrated in figure 3 below.

First 3 cycles of PCR.png
figure 3. First 3 cycles of PCR