The Polymerase Chain Reaction (PCR) is a remarkable technique developed by Kary Mullis in the 1980s (more specifically 1985). Kary Mullis was awarded the Nobel Prize for this work in 1993. The intention of the method is to produce large copies of a single gene which can be used for identification. The production of large quantities of a specific DNA piece is termed amplification.
The process of DNA production this way is an in-vitro (test tube) method.
PCR is one of the modern methods for identifying bacteria species and is especially useful in establishing organisms causing food poisoning for example. The technique has many forensic applications and is now routinely used in cloning and medical diagnostics.
In the process of PCR, an enzyme called DNA polymerase is added to a mixture of DNA bases. Under the right conditions, the enzyme will catalyse the production of many millions of copies of a single gene even from a single starting strand of DNA.
The DNA polymerase is highly thermostable to withstand the temperature changes that take place during the process.
PCR is the starting point or template for DNA sequencing.
The method requires the following:-
- DNA template
- Taq polymerase
- deoxynucleoside triphosphates (dNTPs)
- buffer solutions
- divalent cations such as magnesium ions
The Taq DNA polymerase is perhaps the special catalyst in this method. It is highly thermostable and was isolated from a thermophilic bacterium called thermuys aquaticus which is found in hot springs. This poilymerase has a temperature optimum of 72 centigrade and can survice exposure to temperature w which denature other enzymes. It can survive to 96 centigrade and so will remain active after each of the denaturation steps that it is put through.
In a test tube, DNA is heated to between 92 and 98°C. At these temperatures DNA is denatured and separates from its normal two strand structure into single strands. The DNA is then cooled to between 50 and 65°C. DNA primers are added which bind to target DNA sequences which are of interest to the researcher. Complementary primers are added. These are complementary to the target sequences at the the two ends of the region of DNA to be amplified (increased in number).
The DNA mixture is heated to between 70 and 80°C. The heat tolerant DNA polymerase is added which then replicates a region of DNA to be amplified. In the process, two strands are formed.
The whole cycle of heating and cooling is repeated many times to amplify the production of the target region of DNA. A PCR machine called a thermocycler has been developed which performs this operation routinely.