The polymerase chain reaction, or PCR, was first discovered in 1983 by Kary Mullis. But what is it? It is a method for amplifying small samples of DNA or RNA, creating millions of copies in just a few hours, and has been described as ‘molecular photocopying’. This makes it possible to analyse DNA when only very small amounts are available. Before PCR was widely available, the pieces of DNA had to be cloned into a vector and transferred to bacteria, and this could take weeks – PCR now takes up to a few hours for the same result.
PCR is usually used to amplify stretches of DNA of up to 10 kb (kilo base pairs), though some techniques can work with DNA of up to 40 kb. There three key steps to the PCR, each at a different temperature, and these are repeated in cycles in a PCR machine known as a thermocycler. The process of heating and cooling is called thermal cycling.
Step 1: Denaturing
In the denaturing step (94-98 degrees C for 20-30 seconds), the hydrogen bonds in between the complementary bases and the DNA double helix ‘melts’ and separates into two separate strands. In many PCR thermocyclers, the solutions are held in small plastic tubes in metal blocks, and are heated and cooled by reversing the electrical currents through the block, known as the Peltier effect. However, this can be slow to change temperature and the heating and cooling may not be uniform through the block. Other systems use different technologies – for example, the XXpress thermocycler uses a low-resistance plastic-coated metal sample plate as a resistive element within an electrical circuit to provide heating, along with forced air cooling, to improve speed, accuracy and uniformity.
Step 2: Annealing
At 54-65 degrees C (20-40 seconds), primers (short strands of DNA also known as oligonucleotides) join to the beginning and end of the section of single-stranded DNA to be copied. The two primers are complementary to the 3′ (three prime) ends of the sense and antisense strands of the DNA.
A DNA polymerase enzyme, usually Taq DNA polymerase (from the bacterium Thermus aquaticus) or Pfu DNA polymerase (from the archaeon Pyrococcus furiosus), joins to the DNA. Both of these are heat-stable enzymes, able to cope with the changes of temperature, and this means that the researchers do not need to add new enzymes at the start of each cycle.
Step 3: Extension/elongation
At 72 degrees C, the Taq polymerase reads the two mirror-image strands of DNA and uses them as templates to build two new strands of DNA from a buffer solution containing nucleotides, the building blocks of DNA. The primer matches a stretch of DNA on the section to be copied, and is needed for the enzyme to be able to add new nucleotides, but it also marks out the beginning and end of the piece of DNA to be copied.
The duplicated stretches of DNA each have one old and one new strand of DNA, and can then be used again in the next cycle to create more copies. These cycles are repeated 20 to 40 times, ending up with over one billion copies of the original piece of DNA, known as amplicons.
A final elongation step, at 70-74 degrees C for 5-15 minutes, makes sure that all the single-stranded DNA molecules are completely extended.
Know it all now? Have a go with a virtual PCR lab, and play a PCR game, the Eye of the Donkey!
Suzanne Elvidge is a freelance science, biopharma, business and health writer with more than 20 years of experience. She is editor of Genome Engineering, a blog that monitors the latest developments in genome engineering and that aims to educate (and sometimes to entertain!) and has written for a range of online and print publications including FierceBiomarkers, FierceDrugDelivery, European Life Science, the Journal of Life Sciences (now the Burrill Report), In Vivo, Life Science Leader, Nature Biotechnology, PR Week and Start-Up. She specialises in writing on pharmaceuticals, biotechnology, healthcare, science, lifestyle and green living, but can write on any topic given enough tea and chocolate biscuits. She lives just beyond the neck end of nowhere in the Peak District with her second-hand bookseller husband and two second-hand cats.