Techniques : Polymerase Chain Reaction

The Details:Techniques:
The Polymerase Chain Reaction

Timeline: 1983
PCR Animation

The polymerase chain reaction, or PCR, has been one of the most essential breakthroughs in the history of biotechnology. Developed in 1983 by Kary B. Mullis, the technology allows for quick and inexpensive replication of DNA, empowering scientists to undergo a number of processes, from DNA fingerprinting to mapping the human genome.

PCR is a relatively simple process. DNA is heated to 95 degrees Celsius, until it denatures, or splits, into the separate identical strands of DNA. The mixture is cooled to 55 degrees Celsius and oligonucleotide primers are then added to the mixture. A primer is a section of the DNA used to define the boundaries of the target section, during a step known as annealing. The location at which the primer bonds to the DNA determines the beginning of the section to be replicated. DNA polymerase is added and the mixture is heated to the optimal level of the particular polymerase being used. Polymerase then adds the corresponding base pair, Adenine, Thymine, Cytosine, or Guanine, to the strand, creating two identical copies of the target DNA. This process can then be repeated up to 30 times to create over a billion copies of the sequence.

The high heat involved in denaturing DNA caused problems in the original technique. E. coli DNA polymerase was originally used to reconstruct the DNA. However, the heat also caused the polymerase to split. It also had to be cooled to around 35 degrees Celsius to begin the reaction. Because of these, the polymerase had to be replenished after every step, making the process expensive and significantly slower.

The solution was to use Taq polymerase, derived from Thermus aquaticus, a bacterium that is native to the hot springs of Yellowstone. This was able to withstand the heat (up to 95 degrees Celsius) without denaturing and also required a temperature of around 75 degrees Celsius. This higher temperature allows a more uniform result to be produced. These improvements helped to made PCR cost effective.

For high precision, the Pfu polymerase is used. This is derived from Pyrococcus furiosus and is even better suited to high temperatures(around 100 degrees Celsius) than Taq. It incorporates an additional step known as proofreading. Occasionally, molecules are improperly paired together, for example an A with a G or C. When this occurs, polymerase with the proofreading ability, attempts to remove incorrectly bonded molecules and fix such mistakes.

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