Mullis of cetus corporation in 1983 developed a new technique for rapid replication of a specific DNA fragment in vitro, without the use of bacterial cell. The process is called gene amplification or DNA amplification. The technique was developed on the basis of:
Heat resistant DNA polymerase enzyme is stable at temperature of 90 degree centigrade. It was obtained from bacteria living in hot springs.
Denaturing of DNA at high temperature.
The procedure includes followings steps:
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Cephalus is the largest, and Rhinomugil corsula is found in both fish and brackish waters of Ganga and Mahanadi rivers. The fry of mullets migrate into the brackish water areas and are caught for culture for culture along the tidal creeks and pools.
Catfishes:
Catfishes are not considered for prime importance, but from a large proportion of the total catch. They are relatively cheap and are consumed by poorer A sample of DNA is mixed with all four deoxyribonucleotides, temperature resistant DNA-polymerase enzyme and two short synthetic DNA fragments (oligonucleotides) or primers-p1 and p2.
These primers are complementary to DNA sequence at the 3 and of the DNA to be amplified.
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The oligonucleotides serve as primers to which nucleotides are added during replication.
The mixture is heated to 92-94 degree centigrade. This causes the separation of the two-polynucleotide chains of DNA molecule in the sample.
The mixture is then cooled. The primers get bound to the 3 and of both the strands of the target DNA.
The DNA polymerase binds to the primer and adds nucleotides to the 3 end of the primer.
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As the polymerase extends the primers, it selectively copies the target DNA, forming new complementary DNA strands.
The temperature is raised again causing the separation of newly formed and the parental DNA strands.
The sample is then cooled; to start the polymerase activity i.e. synthetic primers once again bind to the 3 ends of the two copies of the target DNA (i.e., the four DNA strands).
The cycle is repeated over and over again and each time the number of DNA molecules is doubled. Billions of copies of this specific segment of DNA are generated in just few hours.
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Uses of PCR Technique:
The PCR technique helps in amplification of specific DNA fragments.
PCR technique can be used in generating large amount of DNA from minute starting samples i.e., in criminal investigations large amount of DNA is generated from a spot dried blood left on the crimes suspects clothing or even from the DNA present in part of a single hair follicle left on the site of crime.