How does the polymerase chain reaction work?

14 February 2014

Question

Polymerase Chain Reaction - how does it work? I've read a brief explanation
on Wikipedia, but it seemed more to describe the heating cycles scientists go through without, explaining how copies of DNA are produced and amplified.

Answer

Kat - And now it's time for our monthly question. Listener Randy asks, "The Polymerase Chain Reaction - how does it work?" To answer, here's Dr Sarah Hazell, senior science information officer at Cancer Research UK.

Sarah - The Polymerase Chain Reaction, or PCR was dreamed up in the 1980s, primarily by a scientist called Kary Mullis, and is an absolutely fundamental tool in any molecular biology lab. It's a way of making many copies of short stretches of a specific piece of DNA or RNA, like a particular gene, so it can be studied easily.

To do you polymerase chain reaction, your PCR, what you need is your target - such as DNA containing the gene that you're interested in. Then you need two primers - these are very short stretches of DNA that match each end of your gene - and an enzyme called DNA polymerase. DNA polymerase is found in all living cells, as it's the enzyme responsible for copying DNA when a cell divides. For PCR, scientists use a polymerase from a type of bacteria that lives in boiling hot thermal vents in the sea. The reason why they use that enzyme will become clear in a minute.

To do the PCR reaction, you mix your DNA, primers and polymerase, along with the chemicals that the polymerase needs to build new DNA in a very small, thin plastic test tube. Then you put it in a machine called a thermal cycler, which can quickly hit and maintain particular temperatures. To start with, the mixture is heated up to about 95 degrees centigrade - just below boiling - to separate the strands of the DNA. This is why you need the special polymerase, as regular enzymes would be ruined by heating them to this temperature. Then it's cooled down to about 50 to 65 degrees, which allows the short primers to find the particular gene they match. Then the temperature goes up to about 70 degrees or so, which is the temperature the DNA polymerase works best at. It then copies the DNA, starting from the primers. Importantly, the polymerase can only copy DNA where a primer has matched up, so you only get a copy of the particular gene you're interested in. Then the whole thing is heated up to 95 degrees again so the newly-made strands of DNA separate from the template, then the cycle starts again. But this time, because there's a bunch of newly copied DNA that also matches the primers, the primers stick to this too, so copies get made from the copies, as well as the original template. This very quickly builds up to hundreds of thousands of copies over a few cycles - usually about 30 - creating a relatively large amount of DNA in a short time. It's a really clever idea, and one that completely changed molecular biology. No wonder Kary Mullis won a Nobel Prize for it, although it was really a team effort with several people contributing to its development along the way.

Kat - Thanks to Dr Sarah Hazell for that answer. And if you've got any questions about genes, DNA and genetics, just email them to me at genetics@thenakedscientists.com.

Add a comment

CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.