What is PCR?
PCR is shorthand for a simple but very useful procedure in molecular biology called the polymerase chain reaction. It is a technique used to amplify a segment of DNA of interest or produce lots and lots of copies. In other words, PCR enables you to produce millions of copies of a specific DNA sequence from an initially small sample – sometimes even a single copy. It is a crucial process for a range of genetic technologies and, in fact, has enabled the development of a suite of new technologies.
PCR – the polymerase chain reaction explained
This animation explains the three key steps involved in the PCR process. The three steps form a cycle that is repeated many times to produce millions of copies of a specific DNA sequence from a small initial sample.
The mute version of this animation for the activity How does PCR work? can be found here.
How does PCR work?
PCR mimics what happens in cells when DNA is copied (replicated) prior to cell division, but it is carried out in controlled conditions in a laboratory. The machine that is used is simply called a PCR machine or a thermocycler. Test tubes containing the DNA mixture of interest are put into the machine, and the machine changes the temperature to suit each step of the process.
What is PCR?
ESR scientist Dr Brent Gilpin explains how polymerase chain reaction or PCR is able to identify specific bacteria.
Standard ingredients in the mixture are:
the DNA segment of interest
specific primers
heat-resistant DNA polymerase enzyme
the four different types of DNA nucleotides
the salts needed to create a suitable environment for the enzyme to act.
Scientist with a PCR machine
Connie Gilfillan, a PhD student in the Malaghan Institute Immune Cell Biology Programme, uses a PCR machine in her PhD Project 'The role of antigen presenting cell (APC) populations in tumour progression and immunotherapy'.
What is the PCR process?
Step 1: Denaturation
As in DNA replication, the two strands in the DNA double helix need to be separated.
The separation happens by raising the temperature of the mixture, causing the hydrogen bonds between the complementary DNA strands to break. This process is called denaturation.
Step 2: Annealing
Primers bind to the target DNA sequences and initiate polymerisation. This can only occur once the temperature of the solution has been lowered. One primer binds to each strand.
Step 3: Extension
New strands of DNA are made using the original strands as templates. A DNA polymerase enzyme joins free DNA nucleotides together. This enzyme is often Taq polymerase, an enzyme originally isolated from a thermophilic bacteria called Thermus aquaticus. The order in which the free nucleotides are added is determined by the sequence of nucleotides in the original (template) DNA strand.
The result of one cycle of PCR is two double-stranded sequences of target DNA, each containing one newly made strand and one original strand.
The cycle is repeated many times (usually 20–30) as most processes using PCR need large quantities of DNA. It only takes 2–3 hours to get a billion or so copies.
Three steps of PCR
A schematic showing the 3 steps of PCR – denaturation, annealing and extension.
PCR technology is still developing. There is continuing development and refinement of the processes and tools used, allowing the process to be adapted to meet specialist needs. For instance, new methods and refinements are being developed and used, especially when quantification of DNA in a sample is needed. New methods include real-time PCR or quantitative PCR (qPCR) and digital PCR (dPCR). In qPCR, the amplification of DNA is monitored in real time, allowing the quantification of target DNA throughout the process. dPCR is a new, more refined approach that breaks the PCR process up into many smaller steps. It offers increased precision, more reliable measurements and absolute quantification from very small or mixed samples.
PCR: A scientist explains
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.
PCR in the laboratory
In order to prepare a DNA solution for PCR, Tasha needs to add the following ingredients: the DNA molecule to be copied, the enzyme (called Taq polymerase), DNA nucleotides (which will be joined together to make new DNA copies), and RNA primers.
The ingredients are added together and mixed in a buffer solution. The buffer keeps the pH of the whole solution constant (enzymes only work within specific pH ranges).
Nature of science
The development of new technologies, like PCR, enables new discoveries to be made. Other technologies can also be developed. See What is PCR used for? for some examples of how PCR is used and the different types of investigations and processes that are possible because of it.
Related resources
The Hub has a number of related resources, including, PCR in action: Adding genes to cells, and Using gel electrophoresis to check a PCR reaction. There are also many useful applications of PCR technology – see What is PCR used for?
PCR plays a vital role in processing environmental DNA. Learn more about environmental DNA and use this hands-on and feet-on activity to ‘sample’ eDNA in a lake system.
Activity idea
In the activity How does PCR work?, students are asked to view a video and conduct their own research in order to develop an understanding of the polymerase chain reaction process and why it is important.
