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Polymerase Chain Reaction - Essay Example

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This essay will examine the Polymerase Chain Reaction (PCR) technique by taking a closer look at its emergence as the preferred technique for multiplying and amplifying DNA. The advantages of PCR will be highlighted by contrasting the technique with cloning, the components required and the stages of the process.
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Polymerase Chain Reaction
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Introduction This essay will examine the Polymerase Chain Reaction (PCR) technique by taking a closer look at its emergence as the preferred technique for multiplying and amplifying DNA. The advantages of PCR will be highlighted by contrasting the technique with cloning, the components required and the stages of the process.
The Polymerase chain reaction was first introduced Kary Mullis in the 1980's (Bartlett et al 2003). Prior to its use in molecular biology, the amplification of DNA could only be carried out by cloning. PCR allows a "direct amplification without the use of cloning" (Bastianutto et al 2006). Its name was coined from one of its key processes i.e. DNA polymerase. A DNA template is produced and the chain reaction that follows is the concluding part of the process.
Requirements for Polymerase Chain Reaction
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 transformation of a plasmid vector into bacteria that is 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 and
vi. Divalent cations e.g. manganese ions
Stages in Polymerase Chain Reaction
PCR occurs in stages, the Initialization, Denaturation, Annealing, Extension/elongation, Final elongation. The Initialization step involves heating to 94-96C. 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 are fully extended. To check whether the PCR generated the anticipated DNA fragment a gel electrophoresis is carried out.
Polymerase chain reaction helps researchers produce millions of copies of a specific DNA sequence in short time. This efficient process eliminates the need to use bacteria for DNA amplification. Scientists employ PCR in Southern blotting, recombinant DNA technology etc. In microbiology, it is used in diagnosing microbial infections. Law enforcement units also use PCR efficiently in forensics laboratories as only minuscule amounts of original DNA is required. Real-Time PCR can instantly predict DNA present in a sample but also while still in process and rapidly, since it eliminates electrophoresis or other procedure after the DNA amplification reaction.
Bartlett & Stirling (2003). A Short History of the Polymerase Chain Reaction. In: Methods Mol Biol. 226:3-6
Bastianutto, C., Ditta, S. (2006). DNA Fingerprinting/Polymerase Chain Reaction Protocol. Laboratory notes, Seneca College of Applied Arts and Technology, Toronto.
Mackay, I.M. (2007). Real Time PCR in Microbiology: from Diagnosis to Characterization. Queensland: Caister Academic Press. Read More
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