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Describe the process of amplification of "gene of interest" using PCR technique.

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The sequence of steps that take place in a PCR are listed as follows. 

(i) The target DNA is mixed with Taq polymerase, the two oligonucleotide primers and nucleotides. Very small amount of target DNA (just a single molecule) is sufficient as PCR is extremely sensitive. 

(ii) When the mixture is heated to a temperature >90°C, the DNA molecule denatures. The hydrogen bonds that hold together the two polynucleotides of the double helix are broken, so the target DNA becomes denatured to single stranded molecules. 

(iii) The mixture is then cooled down to 50–60 °C where the primers anneal to the DNA molecules at specific positions. The two strands of DNA can also rejoin at this temperature. 

(iv) When the temperature is again raised above 70°C, the DNA polymerase enzyme attaches to one end of each primer and synthesizes new strands of DNA, complementary to the template DNA molecules. 

(v) Now there are four strands of DNA (two original and two new). This process is called extension by which the enzyme extends the primers using the nucleotides provided in the reaction and the genomic DNA as template. When the temperature is then increased back to >90°C, the double-stranded DNA molecules (each of which consists of one strand of the original molecule and one new strand of DNA) denature into single strands. 

(vi) The second cycle of denaturation-annealing-synthesis, at the end of which there are eight DNA strands. This means that after 30 cycles, there will be over 1 billion products derived from each starting molecule.

At the end of a PCR, a sample of the reaction mixture is usually analyzed by agarose gel electrophoresis. The DNA produced should be sufficient for the amplified fragment to be visible as a discrete band after staining with EtBr.

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