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The Three Steps of PCR: Denaturation, Annealing, Extension

Biology · Biotechnology: Principles and Processes · NEET

Each PCR cycle has three steps in a fixed order: (1) Denaturation, the double-stranded DNA is heated so the two strands separate; (2) Annealing, two short primers cool down and bind to the complementary ends of the target region; and (3) Extension, the enzyme Taq DNA polymerase adds nucleotides and builds new strands. Memory hook: "DAE" = Denature, Anneal, Extend, the same order the cell would never let you skip, because you cannot bind primers before the strands open, and you cannot extend before primers bind.
One PCR Cycle: 3 Steps (repeat to amplify)1. Denaturation~94-95 CStrands separate(H-bonds break)2. Annealing~50-55 CPrimers bind ends(orange = primer)3. Extension~72 CTaq builds strandscopies double→→
One PCR cycle runs left to right: denaturation splits the DNA strands at high heat, annealing lets the two primers (orange) bind the ends at a lower temperature, and extension lets Taq polymerase build new strands at ~72 C. Repeating the cycle doubles the DNA each time.

Your doubts, answered

What is the correct order of the three PCR steps?

The fixed order is Denaturation, then Annealing, then Extension. NCERT lists them as (i) Denaturation, (ii) Primer annealing, (iii) Extension of primers. The order is logical: strands must separate first (denaturation) before primers can find them (annealing), and primers must be bound before the polymerase can extend them (extension). NEET has asked this exact sequence many times, so memorise D-A-E.

What actually happens in denaturation?

Denaturation means separating the two DNA strands. The double-stranded DNA is heated to a high temperature (around 94 to 95 degrees Celsius). This heat breaks the hydrogen bonds between the base pairs, so the two strands come apart into single strands. Now each single strand can act as a template for making a new strand.

What happens during annealing, and why is it cooler?

After denaturation the tube is cooled to about 50 to 55 degrees Celsius. At this lower temperature the two chemically made primers base-pair (anneal) to the complementary single-stranded regions that flank the target gene. Cooling is needed because primers can only stick to the template at a lower temperature; if it stayed hot, they would not bind. Primers mark the start point for copying.

What is the extension step and which enzyme works here?

In extension the temperature is raised to about 72 degrees Celsius, the working temperature of Taq DNA polymerase. Starting from each bound primer, Taq polymerase adds free nucleotides (using the provided dNTPs) along the template strand, so a new complementary strand is built. This is where the actual copying of the gene of interest happens.

Why can PCR use these repeated high temperatures without destroying the enzyme?

Because the polymerase used is thermostable Taq DNA polymerase, isolated from the bacterium Thermus aquaticus. A normal enzyme would denature (lose shape) at the high denaturation temperature. Taq stays active through the heat, so the three steps can be repeated many times without adding fresh enzyme each cycle. This is why a thermostable enzyme is essential for PCR.

How does repeating these three steps amplify DNA?

Each full cycle (denaturation, annealing, extension) doubles the number of copies of the target segment, so after n cycles you get about 2 to the power n copies. Repeating the cycle many times gives roughly a billion copies (about 2 raised to 30). This amplified DNA can then be used for cloning, diagnosis, or further study.

⚠️ The NEET trap
✗ Denaturation, Extension, Annealing (or any order that puts extension or annealing first).
✓ Denaturation, Annealing, Extension (D-A-E).
🧠 NTA loves shuffling these three words. Remember the dependency: you cannot anneal a primer to strands that are still stuck together, and you cannot extend a primer that has not yet annealed. So the only possible order is Denature to Anneal to Extend.

Real NEET questions

NEET 2018

The correct order of steps in Polymerase Chain Reaction (PCR) is

A · Denaturation, Extension, Annealing
B · Annealing, Extension, Denaturation
C · Extension, Denaturation, Annealing
D · Denaturation, Annealing, Extension ✓
Solution: Each PCR cycle runs in a fixed order: denaturation (strands separate by heat), then primer annealing (primers bind the complementary ends), then extension of primers by DNA polymerase. Annealing cannot come before strand separation, and extension cannot come before primers bind. NCERT lists the steps in exactly this order, so the answer is Denaturation, Annealing, Extension.
NEET 2021

Which of the following is a correct sequence of steps in a PCR (Polymerase Chain Reaction)?

A · Extension, Denaturation, Annealing
B · Annealing, Denaturation, Extension
C · Denaturation, Annealing, Extension ✓
D · Denaturation, Extension, Annealing
Solution: Each cycle has three steps in fixed order: denaturation (double-stranded DNA separates at high temperature), primer annealing (primers bind complementary regions), and extension of primers by DNA polymerase. This cyclic order amplifies the gene of interest, so the correct sequence is Denaturation, Annealing, Extension.
ReNEET 2026

During PCR, primers bind to the DNA strands in the ______ step.

A · denaturation
B · extension
C · annealing ✓
D · ligation
Solution: PCR has three repeated steps: denaturation (strands separate), annealing (the two primers base-pair to the complementary single strands), and extension (Taq polymerase synthesises new strands). Primers bind during the annealing step, so the answer is annealing.

Solved Biotechnology: Principles and Processes NEET PYQs

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Frequently asked

What are the three steps of PCR in order?

Denaturation, Annealing, and Extension, in that exact order. Remember it as D-A-E.

Which enzyme carries out the extension step in PCR?

Taq DNA polymerase, a thermostable enzyme isolated from the bacterium Thermus aquaticus. It adds nucleotides to extend the primers and stays active despite the high temperatures used.

What are the approximate temperatures for each PCR step?

Denaturation is around 94 to 95 degrees Celsius, annealing is around 50 to 55 degrees Celsius, and extension is around 72 degrees Celsius. NCERT focuses on the sequence and the role of each step more than exact numbers, but the pattern is high, low, medium.

Why are primers needed in PCR?

Primers are two short chemically made DNA pieces that are complementary to the ends of the target region. They mark where copying should begin. DNA polymerase can only add nucleotides to an existing primer, so without primers the extension step cannot start.

How many copies of DNA are made after PCR?

Each cycle doubles the DNA, so after n cycles you get about 2 to the power n copies. After many cycles this reaches roughly a billion copies of the target segment.

Why is a thermostable polymerase used instead of a normal one?

Because PCR repeatedly heats the tube to high temperatures during denaturation. A normal polymerase would be destroyed by that heat, but the thermostable Taq polymerase from Thermus aquaticus stays active, so you do not need to add fresh enzyme every cycle.

Related concepts:
What is PCR (Polymerase Chain Reaction)?What is Taq Polymerase (Thermus aquaticus)?Role of Primers in PCRApplications of PCRProcesses of Recombinant DNA Technology: Overview
Practise Biology for NEET:
Biology MCQs (free, chapter-wise)Biotechnology: Principles and Processes PYQs