PCR: A Revolutionary Invention

Anyone who has worked with DNA in the lab is probably (all too) familiar with a very special reaction, the polymerase chain reaction, or PCR. The technique allows one to make an enormous number of copies of a specified region along any DNA template. If I want to use a protein coding sequence from a particular organism in a synthetic biology application, I can use PCR to copy that sequence alone from DNA extracted from the organism's cells. PCR is also used for so-called DNA fingerprinting in law enforcement applications and for paternity testing. In the medical laboratory PCR can be used to screen for genetic disorders and to identify cancers and pathogens.

Clearly PCR is enormously important for both the scientist and the non-scientist alike. The technique was developed by Kary Mullis in the 1980s, and he received the 1993 Nobel Prize in chemistry as a result. But how does it work? It takes requires several ingredients, the most obvious of which is the DNA template, a sample of DNA that contains the sequence one wishes to copy, or amplify. In theory, this sample could even be a single DNA molecule. Next you need two other pieces of DNA, known as primers. Primers are short pieces of single-stranded DNA (ssDNA) that are complementary to the DNA at the boundaries of the sequence you want to amplify. Sometimes it is useful to choose primers with slight alterations to mutate the template DNA. Finally you need a DNA polymerase, an enzyme that copies DNA, and individual nucleotides-G's, C's, A's, and T's-for the polymerase to incorporate into the DNA copies. The most commonly used variety of DNA polymerase is the enzyme from the thermophilic organism, Thermus aquaticus. This particular DNA polymerase is known as Taq polymerase and is prized for its ability to operate at high temperatures.

Once all of these ingredients are combined, typically in a volume less than a couple drops, they have to be heated and cooled in a particular way. First the double-stranded DNA template has to be separated into two strands. This is how cells copy DNA; since each strand carries all the information to make a duplicate DNA molecule, it is possible to separate the double helix and build two complementary strands to obtain two new DNA molecules just like the original. Cells accomplish this with an enzyme, but in PCR we heat the reaction to near-boiling temperature. This step is called denaturation.

After denaturation the primers have to anneal to the denatured template. The reaction is cooled so that the primers can bind to the template. Next the reaction is heated to around 72° C to optimize the activity of Taq polymerase. An interesting property of DNA polymerase is that it can only copy DNA in a single direction, known as the 5' to 3' direction. These numbers refer to carbon atoms in the sugar component of each DNA base. Now that the Taq polymerase has copied the DNA, the three steps are repeated again: denaturation, annealing, extension; denaturation, annealing extension; etc. The products of one cycle feed into the next one, so the number of DNA molecules grows exponentially. For a perfectly efficient reaction beginning with a single DNA molecule, after 30 cycles you would have 2³⁰, or just over 1 billion, copies. And what's more, this entire process takes at most several hours.

The top image within this post shows a triple water bath setup, where one would be set to the denaturation temperature, the second to annealing, and the third to extension. Doing PCR this way requires shuttling your samples from water bath to water bath at regular intervals. Luckily this is an extremely antiquated way of doing PCR. Nowadays biologists use thermal cyclers like the one shown in the other image above, compact machines that quickly cycle from temperature to temperature based on the user's program.

Molecular biologists have really come to embrace this simple technology. The ability to capture a piece of DNA from any source in massive quantities was revolutionary. Since then the basic reaction has been adapted to suit other applications, such as generating cDNA libraries that reflect gene expression within cells and tissues. There are a number of videos on YouTube that pay homage to PCR, but the most "impassioned" that I've seen is this one. I think PCR more than deserves this praise!

Image Credit: Water baths: Agesworth (via Wikimedia); Thermal cycler: kOchstudiO (via Wikimedia)

References and Further Reading:

Rabinow, P. What is PCR? (1998).