In 1983, Kary Mullis invented the polymerase chain reaction (PCR). It is used to amplify a specific DNA sequence by using a pair of oligonucleotide primers. Each pair is complementary to one end of the target DNA sequence. Therefore, PCR is very useful to generate a DNA fragment required for cloning experiment.
The principle of its work is that at high temperature the hydrogen bonds between the two DNA polymerase using each of the single stranded DNA as template synthesises a new strand.
This event is repeated several times; therefore, original DNA strand is produced in multiple copies. Due to reaction in several cycles it is called PCR. Following are the requirements to operate a PCR:
(i) DNA Template:
Any source that contains one or more target DNA molecules to be amplified can be taken as template.
A pair of oligonucleotides of about 18-30 nucleotides with similar G+C contents act as primers. They direct DNA synthesis towards one another. The primers are designed to anneal on opposite strands of target sequence so that they will be extended towards each other by addition of nucleotides to their 3′ ends.
The most common enzyme used in PCR is a thermostable enzyme called Taq polymerase. It is isolated from a thermostable bacterium called Thermus aquaticus. It survives at 95°C for 1-2 minutes and has a half life for more than 2 hours at this temperature. The other thermostable polymerase can also be used in PCR.
1. Working Mechanism of PCR :
The action of PCR involves several cycles. However, there are three steps in one amplification cycles e.g denaturation (melting), annealing and polymerisation (extension).
The two strands of DNA are separated by applying a high temperature (95°C). After separation each strand acts as template for DNA synthesis.
(ii) Primer Annealing:
Since nucleotide sequence of each of oligonucleotide primer is complementary to 3′ end of single stranded template, the primers anneals (hybridizes) the each template. Annealing is done at low temperature based on length and sequence of the primers.
The annealing temperature varies, but the too low temperature favours mispairing. The annealing temperature (in °C) can be calculated using the formula: T = 2(AT) + 4(G + C).
(iii) Extension (Polymerization):
It is the final step of amplification cycle. In the presence of Mg++ and dNTPs (deoxynucleotide triphosphates e.g. dATP, dGTP, dCTP, dTTP), Taq DNA polymerase (at 70°C for 60-90 seconds) synthesis through onward extension of primer in direction on single DNA template.
The concentration of Mg is maintained between 1 and 4 mM. Thus in the first step the target DNA is copied from the primer sites for various distances until the start of second cycle.
The second cycle starts and doubles the DNA molecules synthesised in the first cycle. The second cycle is started with heating of double stranded DNA to result in single stranded DNA.
Each single stranded DNA again acts as template i.e. DNA molecules polymerised in first cycle act as template in second cycle. Following the above events all the single stranded DNA molecules of second cycle are converted into the double stranded DNA.
Then third and onward cycles are repeated in the same ways to get more DNA products. Always after n number of cycles, 2″ molecules of DNA are generated using single stranded DNA as template.
2. Application of PCR :
After the discovery of PCR, the modern biology has been revolutionised in each and every aspects. Some of the areas of application of PCR have briefly been discussed herewith.
(a) Diagnosis of Pathogens:
There are several pathogens that grow slowly. Therefore, their cells are found less in number in the infected cells/tissues. It is difficult to culture them on artificial medium.
Hence, for their diagnosis PCR-based assays have been developed. These detect the presence of certain specific sequences of the pathogens present in the infected cells/tissues. Besides, it is useful in detection of viral infection before they cause symptoms or serious diseases.
(b) Diagnosis of Specific Mutation:
In humans there are thousands of genetic diseases. Mutations are also related to genetic diseases. Presence of faulty DNA sequence can be detected before establishment of disease.
(c) In Prenatal Diagnosis:
It is useful in prenatal diagnosis of several genetic diseases. If the genetic diseases are not curable, it is recommended to go for abortion.
(d) DNA Fingerprinting:
In recent years DNA fingerprinting is more successfully used in forensic science to search out criminals, rapists, solving disputed parentage and uniting the lost children to their parents or relatives by confirming their identity.
This is done through making link between the DNA recovered from samples of blood, semen, hairs, etc. at the spot of crime and the DNA of suspected individuals or between child and his/her parents/relatives.
(e) In Research:
In addition, DNA fingerprinting of new microorganisms isolated from various extreme environment (soil, water, sediments, air, extreme habitats, etc.) is also carried out to confirm their identity by comparing with the DNA sequences of known microorganisms.
(f) In Molecular Archaeology (Palaentology):
PCR has been used to clone the DNA fragments from the mummified remains of humans and extinct animals such as the woolly mammoth. DNA from buried humans has been amplified and used to trace the human migration that occurred in ancient time.