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Polymerase Chain Reaction (PCR) is an important technique used in Recombinant DNA technology for amplification of desired DNA molecule or gene of interest.
As the cell amplifies DNA by replication in vivo, PCR is a method of amplifying DNA in vitro (in a test tube). PCR uses the same principle of amplification as cell uses for replication.
- PCR stands for Polymerase Chain Reaction.
- Kary Mullis invented the Polymerase Chain Reaction (PCR) in 1985.
- PCR can amplify as minute as picogram (10-12) quantity of DNA.
- In Recombinant DNA technology, PCR is used to amplify (to make billions of copies) the gene of interest/ DNA fragment which is separated and isolated by Agarose gel electrophoresis.
- As the cell uses template and primers for DNA replication, PCR uses the same. Appropriate primers should be available for DNA fragment to be amplified.
- PCR requires the following components:
- DNA fragment to be amplified / Gene of interest (template DNA).
- Two primers (one for each strand). Primers are small chemically synthesized oligonucleotides that are complementary to the 3’ ends of both strands of DNA fragment.
- Enzyme DNA polymerase: It extends the primers using the nucleotides provided in the reaction mixture. DNA Polymerase used in PCR is Taq DNA Polymerase, isolated from thermophilic bacterium, Thermus aquaticus. Taq DNA Polymerase is thermostable DNA Polymerase and remain stable at temperature of around 74 ºC, that’s why it is used in PCR. Normal DNA Polymerase enzymes break down at such high temperature.
- PCR is carried out in a instrument known as Thermal Cycler. Single test tube containing mixture of DNA fragment and a set of reagents is placed in thermal cycler equipment that enables the mixture to be incubated at a series of temperatures that are varied in a preprogrammed manner.
- Basic steps in a PCR experiment are as follows:
- Denaturation of template DNA at 94 ºC.
- Annealing of oligonucleotide primers at 50-60 ºC.
- Extension of Primers/ Synthesis of new DNA at 74 ºC.
- Repetition of cycle 25-30 times
1) Denaturation: Mixture is heated to 94°C. Hydrogen bonds holding the two strands of double-stranded DNA molecule are broken, which causes denaturation of DNA template.
2) Primer Annealing: Mixture is cooled down to 50–60°C. At this temperature, denatured DNA strands can again join back together but usually do not, because mixture has large amount of oligonucleotide primers, which get anneal to the DNA molecules at specific positions.
3) Extension of Primers/ Synthesis: Now temperature is raised to 74°C, which is a good working temperature for the Taq DNA polymerase that is present in the mixture. Taq DNA polymerase attaches to one end of each primer and synthesizes new strands of DNA, complementary to the template DNA. This step produces four stands of DNA (i.e. 2 DNA molecules) after completion.
4) Repeat the cycle: Temperature is again increased back to 94°C. At this temperature DNA molecules each containing one strand of the original molecule and one new strand of DNA, denature into single strands. Now there are 4 strands of DNA instead of 2, which were present in starting. This begins a second cycle of denaturation–annealing–synthesis at the end of which there are 8 DNA strands.
In this manner repeating the cycle 25-30 times, billions of copy of DNA molecule between the primers can be produced in just a few hours. This amplified DNA can then be inserted into a vector to create recombinant DNA and be cloned in host cells.