Polymerase chain reaction is a molecular biology technique that is used to amplify DNA creating several thousands to millions of copies. It was first developed by Kary Mullis in 1983, for which he was awarded the Nobel Prize. Today, it is an indispensible part of DNA analysis and molecular biology experiments.
The principle behind PCR is known as thermal cycling. The DNA sample containing the target sequence undergoes multiple cycles of heating and cooling that promote melting of the double helix and consequent replication of the DNA. Primer sequences that are complimentary to the target DNA are used for the initiation of the replication. The enzyme DNA polymerase that helps in the replication is also added in the reaction mixture. Since there is repeated heating and cooling of the reaction mixture, the polymerase has to be heat-stable in nature. Most labs use Taq polymerase which is isolated from Thermus aquaticus. As and when new strands are created, they act as template for the replication in the next cycle. This process goes on for about two house. This chain reaction is responsible for the amplification process that results in the high copy number in the PCR product.
The Taq polymerase uses the nucleotides present in the reaction mixture, along with the primers and the DNA templates, creating new strands. The thermal cycling process is applied so that the heat can melt the DNA double helix into two separate strands. The cooler temperature then facilitates the replication process and the The amplification of the DNA is so highly selective due to the presence of the specific primers that amplify a very specific part of the DNA strand.
PCR can be used to amplify DNA fragments that are ten to upto fourty kilo base pairs in size.
To run a PCR, the following reagents and components are required in what forms the 'reaction mixture':
-A DNA template that carries the target DNA that needs to be amplified
-Primers that are complementary to the 3' ends of the two strands (the sense strand and the anti-sense strand) of the target DNA
-A DNA polymerase with the right optimum temperature (Taq polymerase is commonly used)
-dNTPs that are used to build the new strands. These include dATP, dGTP, dCTP and dTTP.
-A buffer that provides the optimum conditions for amplification to occur
-Magnesium cations or Manganese cations
The total reaction mixture is around 10-200 microlitres. It is placed in thin reaction vials that allow fast conduction of the temperature changes to the reaction mixture. The machine usually has a heated lid to prevent condensation within the reaction vials. If heated lid is not present, a drop of mineral oil is added on top of the reaction mixture for the same purpose.
The PCR products are tested for accuracy. Agarose gel electrophoresis is performed with a ladder of known size. The success of the amplification process depends on the purity of the reaction sample. Any contamination can cause amplification of the wrong targets. To avoid this, preparation of reaction mixture is done in sterile conditions or in conditions where no contamination is possible. Adequate precautions are used to maintain the integrity of DNA prior to starting the thermal cycling process. The reagents are added in exact amounts to obtain ideal results.
Applications of PCR:
It is used for amplification of DNA for cloning purposes and for creating hybridization probes. It can be used to identify DNA fingerprints and perform paternity testing. DNA sequencing and rDNA processes may require amplified product of sample DNA that can be provided by PCR. Screening of microbial colonies can be performed by using the appropriate primer for the vector. It can be used to establish evolutionary connection between related organisms. Forensic analysis can be performed with minute quantities of DNA. Identification of specific strains of microorganisms can be performed by PCR by using primers for known segments of DNA. Viral DNA can be detected upon infection rather than after commencement of the disease symptoms. This can save valuable time which can be devoted to treatment.
There are some variations of the PCR technique like hot-start PCR, inverse PCR, multi primer PCR, multiplex PCR, Nested PCR, RT PCR and several others.
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