Rights: The University of Waikato Published 21 November 2007 Download

Clare Eagleton, a scientist at Genesis R&D, explains how multiple copies of DNA are made during a PCR reaction. The reaction involves a repeating cycle of denaturation (the two strands in the DNA double helix are separated), annealing (DNA primers bind) and extension (new DNA is made). The new DNA is complementary to the DNA that is being sequenced. Each time the cycle is repeated, the number of DNA copies is doubled. In the first cycle, one DNA double helix is copied to form two double helices. These two helices are then copied, resulting in four copies. The result at the end of the third cycle is eight copies of the DNA. Sixteen copies after four cycles, and so on.


Dr Clare Eagleton (Genesis R&D)

It is quite abstract, because you load all of the reagents into the tube and then you stick it into the machine. So I am going to explain here what happens when you do that.

The first thing is that you have double stranded DNA, and the first thing that you do is the machine heats up to 94 degrees and what that does is it actually denatures the double strand. If you imagine it being coiled, it uncoils and separates into two strands, and that process is called denaturation.

Once it has done that, the primers come and anneal to the DNA. This is called annealing and at this stage, the temperature has dropped now to 55 degrees to allow these to bind.

The next stage is extension and the temperature raises to 72 degrees and the Taq polymerase allows the second strand to be formed.

Your final product at the end of that cycle is actually two complementary strands of DNA being made. Now you have two copies of what you started with.

Now in PCR, this machine is designed to basically take this cycle of denaturing and annealing and extension, and doing it in this case, thirty times. Each time you end up with an exponential amount of DNA.