9.3.3. Amplification of Gene of Interest using PCR

PCR stands for Polymerase Chain Reaction. In this reaction, multiple copies of the gene (or DNA) of interest is synthesised in vitro using two sets of primers (small chemically synthesised oligonucleotides that are complementary to the regions of DNA) and the enzyme DNA polymerase.

PCR is a powerful technique used in molecular biology to amplify specific DNA sequences, enabling scientists to create many copies of a particular gene. This process begins with two short sequences of nucleotides known as primers, which are designed to match exactly with the ends of the target DNA sequence. The enzyme DNA polymerase then extends these primers, synthesizing new DNA strands complementary to the target template. This allows for exponential amplification of the specific DNA segment.

Imagine you are trying to make copies of a favorite drawing. You can trace the outline with a pencil (the primer) and then use a marker (the polymerase) to fill in the details, creating a perfect replica of your drawing. With PCR, this tracing and filling process is repeated multiple times, resulting in many copies of the original drawing.

The enzyme extends the primers using the nucleotides provided in the reaction and the genomic DNA as template. If the process of replication of DNA is repeated many times, the segment of DNA can be amplified to approximately billion times, i.e., 1 billion copies are made.

In a typical PCR cycle, there are three main steps: denaturation, annealing, and extension. Denaturation involves heating the reaction to separate the two strands of the DNA. During annealing, the temperature is lowered to allow the primers to attach to their complementary sequences on the single-stranded DNA. Finally, in the extension step, the temperature is raised again, allowing DNA polymerase to add nucleotides and synthesize a new strand of DNA from the template. This cycle is repeated around 25 to 35 times, leading to an exponential increase in the amount of the target DNA.

Think of PCR like a photocopier that can repeatedly make copies of a document. Each cycle of PCR is like one pass of the copier; after each pass, the number of copies doubles. After several passes, you end up with an enormous stack of copies from the original document.

Such repeated amplification is achieved by the use of a thermostable DNA polymerase (isolated from a bacterium, Thermus aquaticus), which remain active during the high temperature induced denaturation of double stranded DNA.

The DNA polymerase used in PCR is not like regular enzymes; it needs to withstand high temperatures used during the denaturation phase. Thermus aquaticus, a bacterium that thrives in hot springs, produces a DNA polymerase known as Taq polymerase that remains functional even at extreme temperatures. This stability allows it to catalyze DNA synthesis throughout the PCR cycles without needing to be replaced.

Think of Taq polymerase as a marathon runner who is trained to run in the heat of a desert. While other runners might tire and need to stop for water, Taq keeps going, consistently performing its job without pause, allowing PCR to produce many copies of DNA continuously.

The amplified fragment if desired can now be used to ligate with a vector for further cloning.

Once the target DNA has been sufficiently amplified, the next step involves acquiring a cloning vector, which is a DNA molecule that can carry the amplified DNA into a host cell. The amplified DNA is combined with the vector DNA and treated with DNA ligase, an enzyme that helps to join the DNA ends together, forming recombinant DNA that can be introduced into host organisms for expression or further study.

Imagine you have built a beautiful model of a car out of LEGO bricks (the amplified DNA). Now, you want to add this model into a playset (the vector) that allows for more creative expansions (cloning). You carefully attach your car model to the playset with strong glue (DNA ligase), allowing it to become part of a larger, interactive environment.

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