Polymerase Chain Reaction - Essay Example

A major requirement of recombinant DNA techniques is the “availability of large quantities of specific DNA segment” (Bastianutto et al 2006). Cloning which was the preferred method involves the transformation of a plasmid vector into bacteria that are then cultured. The cloning process is not as efficient as PCR in terms of amplification of DNA. In addition, PCR can allow the generation of millions of copies of DNA from a single or few pieces. PCR mostly uses DNA as a target instead of RNA.

This target can be a gene or a non-coding sequence.  A successful PCR technique requires the following components and reagents:i. DNA Template containing the target to be amplified.ii. a pair of complementary primers at 5’ prime and 3’ ends.iii. an enzyme: Taq polymerase.iv. Deoxynucleoside triphosphates (dNTPs) building blocks of the new strands.v. A buffer to provide a DNA polymerase suitable balanced chemical environment andvi. Divalent cations e.g. manganese ionsPCR occurs in stages, Initialization, Denaturation, Annealing, Extension/elongation, Final elongation.

The Initialization step involves heating to 94-96°C. After the heating stage, Denaturation produces a single DNA strand. This is caused by breaking the hydrogen bonds between DNA strands, thus causing the melting of the DNA template and primers. Temperature is lowered during the annealing step. Here primers are bound to the single-stranded DNA template by the polymerase. At the Extension/elongation, the DNA polymerase produces a new DNA strand by adding dNTPs. The last PCR cycle is the Final elongation, the step is done to ensure that all single-stranded DNA is fully extended.

To check whether the PCR generated the anticipated DNA fragment gel electrophoresis is carried out. Polymerase chain reaction helps researchers produce millions of copies of a specific DNA sequence in a short time. This efficient process eliminates the need to use bacteria for DNA amplification. Scientists employ PCR in Southern blotting, recombinant DNA technology, etc.