Polymerase chain reaction or PCR in biotechnology is a technique of Amplifying single piece of Deoxyribose nucleic acid (DNA), into millions copies of DNA sequence. This technique is dependent on thermal cycling in which, repeated heating and cooling is done for melting of DNA and for enzymatic replication of the DNA. The two key elements for these reactions are fragments of DNA called as primers and enzyme DNA polymerase. These two elements reaction help in repeated and selective amplification of the test sample DNA.
For any DNA to replicate template is required and in PCR technique, DNA generated is acting as template for the replication. This set a continuous reaction in chain .This way, the DNA template is amplified to the level that is required for complete identification of DNA and its further usage. Genetic manipulation is done extensively with specific modification in PCR. The reaction basically requires DNA fragments or DNA piece and its suitable enzyme. The enzyme in this case should be heat stable, as this is cycle of thermal reaction. Certain bacteria produce such enzymes which can remain stable at even high temperature and does not lose their enzymatic properties. One such known bacteria is Thermus aquaticus , which produces DNA polymerase enzyme Taq polymerase. The nucleotides which are building block of DNA are used to form new strand of DNA. The single stand of DNA is used as template while primer is DNA oligonucleotide. This reaction of various bio-molecules in complex initiates synthesis of DNA.
This technique is extensively used in this field of medical and biological research labs. Well know applications of these techniques are DNA cloning and sequencing, gene functional analysis, hereditary diseases diagnosis, genetic finger printing (forensic studies), diagnosis of various infectious diseases and timely diagnosis of malignant like leukemia and lymphomas and thus helps in cancer research. In genomic DNA samples PCR is employed to detect translocation-specific cancer cells at 10,000 fold higher sensitivity than ordinary methods etc. This technique was so important and revolutionary that Kary Mullis, who developed it was awarded with Nobel prize in 1993.
In detail, PCR techniques consist of around 30 repeated cycles of temperature changes. Initialization step consist holding reaction of DNA piece, enzyme polymerase and primers at 94-96 °C temperature for 2-9 minutes. Further to this, denaturation of DNA strand takes place, in which DNA melts into single stranded DNA molecules. Next to this, annealing is done. In annealing step, temperature of reaction is being down to around 55 °C for 20-40 seconds. DNA-DNA hydrogen bonds are formed which are stable in nature. To the hybrid of primer-template, polymerase binds and results in synthesis of DNA. After this initiation, elongation of DNA is done. The temperature depends on the enzyme being used considering its thermal stability. New strand synthesis of DNA takes place which is complementary of the template. The direction of this extension is in 5' to 3' direction of DNA, thus 5'-phosphate group is condensed with 3'hydroxyl .This innovative work by Kary Mullis has been a revolution in the field of biotechnology and such excellence always results in awards not less than Nobel prizes!
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