What is PCR? Explain the steps involved in amplification of Gene of interest using PCR.

Explanation

(A) Amplification of Gene of interest using PCR : Amplification of gene is a process of making many copies of a gene". It is achieved by using a technique called Polymerase Chain Reaction (PCR).
Requirements:
• A DNA containing the desired segment to be amplified . Two nucleotide primers (about 20 base long) specific i.e. complementary to the two 3 - ends of the desired segment.
• The four deoxynucleoside triphosphate i.e. TPP (Thymidine triphosphate) dCTP (Deoxycytidine triphosphate) DATP (deoxyadenosine triphosphate) and dGTP (deoxyguanosine triphosphate)
• Thermostable DNA polymerase i.e., Taq DNA polymerase.
Procedure of PCR : 1. The DNA from the desired segment to be amplified, an excess of the two primer molecules, the four deoxyribose triphosphates and DNA polymerase are mixed together in a reaction mixture in an eppendorf tube with sufficient quantities of `Mg^(++)` The eppendorf tube is placed in the PCR unit and the following operations are performed sequentially.
2. Denaturation : The reaction mixture is first subjected to a temperature between `90-98^(@)C` `(commonly 94^(@))`. It results in the separation of two strands of DNA due to the breakage of hydrogen bonds. This is called denaturation. Each strand of DNA acts as a template strand for DNA synthesis. The duration of this step in the first cycle of PCR is usually 2 minutes at` 94^(@)`
3. Annealing (anneal=join): The mixture is now cooled to a low temperature `(40-60^(@)C)`. During this step, two oligonucleotide primers, complementary to a region of DNA, anneal (hybridize) one to each 3 end of DNA strand. The duration of annealing step is usually one minute during the first as  well as the subsequent cycles of PCR.
4. Primer extension : During this step, the enzyme taq DNA polymerase extends the primers using nucleotides and DNA templates. The two primers extend towards each other in order to get two new strands of DNA (at 5) end. The duration of primer extension is usually 2 minutes at `72^(@)`The amplified fragment, if required, can now be used to ligate with a vector for further cloning. The taq DNA polymerase remains active, during the high temperature induced denaturation of double stranded DNA