Q.Identify and explain steps 'A', 'B' and 'C' in the PCR diagram given below.

Let’s look at the three steps of PCR in the order they appear in the NCERT diagram.

Step A is Denaturation. The reaction mixture is heated to a high temperature (the NCERT text does not give an exact figure). This high temperature breaks the hydrogen bonds between the two strands of the target DNA, separating them into single strands. Each single strand will now act as a template for the next step.

Step B is Annealing. The mixture is cooled so the primers can bind. At this lower temperature, the two short synthetic oligonucleotide primers (one complementary to each strand) bind to their specific target sequences on the single-stranded DNA. This sets the boundaries for the region to be amplified. …

Polymerase Chain Reaction, or PCR, is a technique that copies a specific segment of DNA millions of times in a few hours. It was developed by Kary Mullis in the 1980s and has become a cornerstone of molecular biology, used in everything from disease diagnosis to forensic science. The process relies on a heat-stable DNA polymerase (usually Taq polymerase) and a thermal cycler that precisely changes the temperature of the reaction mixture. The diagram you are referring to shows the three repeating steps that make up one PCR cycle.

Let us walk through each step as labelled in the diagram.

Step A: Denaturation — This is the first step, where the reaction mixture is heated to a high temperature (the NCERT text does not give an exact figure). At this high temperature, the hydrogen bonds between the two strands of the target DNA break apart. The double-stranded DNA molecule unwinds and separates into two single strands. Think of it like unzipping a zipper — each single strand now serves as a template for the next step. This step is essential because DNA polymerase can only work on single-stranded DNA.

Step B: Annealing — The temperature is now lowered so the primers can bind. This allows the two short, synthetic DNA primers (which were added to the reaction mixture) to bind, or "anneal," to their complementary sequences on each single-stranded template. Each primer is designed to flank the target region — one binds to the start of the sequence on one strand, and the other binds to the end of the sequence on the opposite strand. The primers act as starting points for DNA synthesis. The exact annealing temperature is chosen to ensure specific binding; too low, and primers may stick to the wrong places; too high, and they may not bind at all. …

Method 2 -- read the steps off a temperature-vs-time profile

A PCR reaction is easiest to identify from the shape of its temperature profile over one cycle, rather than from a written description:

• A high-temperature plateau in the profile is always denaturation -- this is the only step hot enough to break the hydrogen bonds holding the two DNA strands together.
• A lower-temperature plateau, coming right after the high-temperature step, is always annealing -- primers can only bind once strands have separated, and this is the temperature window where primer-template base pairing is both possible and specific. …