Polymerase Chain Reaction (PCR)
The polymerase chain reaction, or PCR, is a method used in molecular biology and genetics laboratories to amplify specific regions of DNA. The ability to create millions of artificial copies of a specific piece of DNA in a test tube, from tiny amounts of starting material, makes PCR one of the most widely used laboratory techniques with applications in almost every area of molecular biology and genetics, including medicaldiagnostics, forensic science and evolutionary biology. PCR was developed by Kary Mullis in 1983 when he was working on DNA synthesis at Cetus Corporation in California. He was awarded the Nobel Prize in chemistry in 1993 for his invention.
Components of a PCR reaction
A successful PCR reaction requires the following components
- A DNA template that contains the target sequence of interest
- DNA polymerase – this is the enzyme that synthesizes new strands of DNA
- Nucleotides – these are the individual building blocks of DNA
- A pair of DNA primers – these are short fragments of DNA (around 20-30 nucleotides long) that are complementary to the target sequence of interest. One primer flanks the left hand end of the target region, while the other binds to the opposite DNA strand and flanks the right hand end.
- Buffers containing magnesium ions – these provide the correct chemical conditions for the polymerase to work.
How Does PCR Work?
PCR is a three-step, cyclic process, where new strands of DNA are created through successive rounds of denaturation, annealing and extension using the enzyme DNA polymerase.
Firstly a denaturation step is performed where the DNA is heated to around 95°C to separate out the two strands. Secondly, an annealing step takes place where the temperature is lowered to around 50-60°C to allow the DNA primers to bind to the separated strands. In the third step, extension, the DNA polymerase synthesizes a new strand of DNA by adding nucleotides to one end of each DNA primer. This makes a copy of the region of DNA in between the two primers. The PCR reaction cycles between these three steps 30-40 times. At every cycle, the amount of target sequence is doubled, so the reaction proceeds in an exponential manner until components like primers and nucleotides begin to be used up and the polymerase begins to lose its activity. For an animation of this process, see here.
Two factors were crucial in enabling PCR to be developed as an efficient laboratory technique: the discovery of thermostable DNA polymerases, and the invention of the automated thermal cycler. Thermostable DNA polymerases were originally isolated from the thermophilic bacterium Thermus aquaticus, found in the hotsprings of Yellowstone National Park. Unlike regular DNA polymerases, T. aquaticus or Taq polymerases are able to withstand being heated to 95°C meaning they do not need to be added at each new cycle of the PCR. Using Taq polymerase, the extension step can be performed at 72°C.
Automated thermal cyclers enable efficient cycling of temperatures between the denaturation, annealing and extension steps. When PCR was first developed, researchers used waterbaths set at the 3 different temperatures and had to manually move tubes between baths, making PCR a time-consuming, labour intensive task. Today’s modern thermocyclers comprise metal blocks where tubes containing the PCR reaction can be inserted. The thermocycler is pre-programmed to move between temperatures for highly accurate and fast cycling, enabling most PCR reactions to be completed in less than 2 hours.
Uses of PCR
Using PCR, researchers are able to efficiently isolate and amplify selected regions of DNA from very small amounts of starting material. These regions may be individual genes, regions of DNA that regulate how genes are switched on or off, or repetitive regions of DNA that enable individuals to be identified. This ability to pick out and amplify a specific region from a DNA template has enabled the development of many sensitive diagnostic tests that would otherwise be extremely difficult, expensive and time-consuming to perform.
Applications of PCR include:
- Isolation of individual genes to study their function
- Studies of populations and species: By amplifying genetic markers that vary among populations or species, researchers are able to investigate relationships between individuals, estimate population sizes, study how populations are formed and trace evolutionary relationships between species.
- Forensic analyses: The highly sensitive nature of PCR means that DNA can be amplified from tiny amounts of starting material, such as individual hairs or trace amounts of blood. This, combined with the fact that many genetic markers are variable enough to be able to identify individuals, makes PCR a valuable tool in forensic science.
- Disease diagnostics. PCR can enable early diagnosis of diseases by detecting DNA sequences specific to infectious disease organisms, or mutations which are diagnostic of cancer cells or inherited diseases.
- Quantification of DNA. This uses a variation on the basic PCR method called “real-time” PCR, which makes uses of the exponential nature of the PCR reaction. The accumulation of DNA product at each round of the reaction is measured, enabling researchers to extrapolate back to determine the amount of starting material.