Polymerase Chain Reaction (PCR)

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PCR (Polymerase chain reaction)
Polymerase chain reaction (PCR) is a laboratory method that is used to form number of copies of a specific region of DNA interested in. The principle of the polymerase chain reaction is based on the natural replication process of DNA and complementary nucleic acid hybridization. The goal of the PCR is to produce required amount of the target DNA region that is be analyzed. PCR amplified DNA is sent for sequencing and visualized by gel electrophoresis. It also can be cloned into plasmids. PCR is used in many fields of medicine, molecular biology research, medical diagnostics, and ecology. It is fast and inexpensive technique used to amplify DNA and RNA fragments by 107 times. The polymerase chain reaction was
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The components are inserted in to a tube with cofactors (Mg) and the enzyme. The DNA is synthesized through repeated cycles of heating and cooling.
The sample used in PCR should isolate the DNA before processing it. Therefore DNA extracted from the sample manually or using instruments like COBAS Ampliprep. (National Human Genome Research Institute, 2015) The sample used in PCR should be free from DNA contaminations.
The basic steps of PCR are:
1. Denaturation – the main purpose of this step to separate or to denature the DNA strands by heating the reaction strongly. This provides single-stranded template for the next step. This is carried at a temperature of 900c (1940F) to break the bonds between nucleotides forming DNA code ensuring the accessibility of the gene of
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Extension - temperatures of the reaction is raised to extend the primers and synthesize new strands of DNA by Taq polymerase. This is carried at a temperature of 720c (161.50F). The cycle is repeated by denaturing and synthesizing the new DNA strands with multiple copies.

PCR cycle takes few hours if the cycle is automated. The thermocycler is a directed machine to program the alteration of the temperature in the reaction for denaturing and synthesizing. The temperature regulation is essential to optimize the results. The length of the DNA region to be copied depends on the cycle repeats (25 - 40) and time (2 - 4 hours). The new DNA made in one round can serve as a template in the next round of DNA synthesis. The number of DNA molecules double in each cycle due to the multiple copies of primers and Taq polymerase. The copy number is the number of amplified DNA copies produced in the PCR. It is calculated by multiplying the Avogadro number by the moles of DNA in the input.

Gel electrophoresis is used to visualize the results obtained in the PCR reaction. In gel electrophoresis the DNA fragments are separated in the gel matrix through an electric current. The separation is carried out according to the size and charge. The sample can be detected by the DNA ladder included. (Kumar,

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