Q.What is the Polymerase Chain Reaction (PCR)? Name the three main steps of one PCR cycle and state the approximate temperature at which each step is carried out.
Concept understanding — Polymerase Chain Reaction (PCR)
PCR is a common laboratory technique used to make millions of copies of a chosen region of DNA. In recombinant DNA technology, it serves as the alternative route to vector-based cloning wherever no vector is actually used: instead of inserting the gene of interest into a self-replicating vector and propagating it inside a host cell, PCR directly amplifies that gene of interest in vitro, after which the resulting multiple copies are delivered into the host cell's protoplast either by direct injection or by the biolistic (shot-gun) particle-bombardment method.
[!TLDR] PCR = denaturation (~94-96C) -> annealing (~50-65C) -> extension (~72C), repeated to exponentially amplify a target DNA segment.
[!ANSWER] PCR (Polymerase Chain Reaction) amplifies a chosen DNA segment using repeated cycles of: (1) denaturation, around 94-96 degrees C, separating the double-stranded template into single strands; (2) annealing, around 50-65 degrees C, allowing the two primers to bind their complementary sequences flanking the target; (3) extension, around 72 degrees C, where Taq polymerase synthesises new complementary DNA strands from each primer.
The Polymerase Chain Reaction amplifies a specific segment of DNA outside a living cell, using a repeating three-step temperature cycle. In the denaturation step, the reaction is heated to a high temperature, typically 94 to 96 degrees Celsius, which breaks the hydrogen bonds holding the double-stranded template together, separating it into two single strands.
In the annealing step, the temperature is lowered, usually to somewhere between 50 and 65 degrees Celsius (the exact value depends on the specific primers being used), allowing the two short, single-stranded DNA primers to find and bind, through complementary base-pairing, to their matching sequences flanking the target region -- one primer on each of the two separated template strands.
In the extension (elongation) step, the temperature is raised again, typically to around 72 degrees Celsius, the optimal working temperature for Taq polymerase, which then extends each primer by adding complementary nucleotides one at a time, synthesising a new complementary strand and effectively copying the target region.
[!ANSWER] One full PCR cycle is denaturation (~94-96 degrees C) -> annealing (~50-65 degrees C) -> extension (~72 degrees C); because the products of each cycle serve as templates for the next, repeating this cycle roughly doubles the amount of target DNA each time, producing exponential amplification.
State the three steps in strict order with their approximate temperatures, and note that the cycle repeats.
Do not reverse the temperature order -- denaturation is the HIGHEST temperature step, extension is intermediate, and annealing is the LOWEST of the three.
Showing the 12 most recent of 24 on this concept.
- CBSE 2026Set V11 markMCQQ.Which of the following is the correct representation of primer annealing step in PCR?(a) 5'——— 3' / 5'——— 3' (short primer strands shown near 5' ends)(b) 3'——— 5' / 3'——— 5' (short primer strands shown)(c) 5'——— 3' / 3'——— 5' (short primer strands shown)(d) 3'——— 5' / 5'——— 3' (short primer strands shown)
›Reveal solutionSolution
The two separated template strands are antiparallel (one 5'→3', the other 3'→5'), and the two primers anneal to them — represented by option (c).
In PCR, after denaturation the double-stranded DNA separates into two antiparallel single strands: one runs 5'→3' and its complement runs 3'→5'. During the annealing step, the two short primers base-pair (anneal) to these two complementary strands. Only option (c), showing one strand 5'——3' and the other 3'——5' with primers bound, correctly represents the antiparallel templates.
✓Final answer(c) 5'——— 3' / 3'——— 5' (antiparallel template strands with primers annealed)
- CBSE 2026Set A1 markMCQQ.Themrostable DNA polymerase used in PCR is isolated from which bacteria?(a) Thermus aquaticus(b) E. coli(c) Salmonella(d) Eubacteria
›Reveal solutionSolution
Taq polymerase comes from Thermus aquaticus; the correct option is (a).
PCR (Polymerase Chain Reaction) involves repeated cycles of denaturation at about 94 to 95 degrees Celsius. An ordinary DNA polymerase would be destroyed at these high temperatures. Therefore a thermostable DNA polymerase, called Taq polymerase, is used. It is isolated from the thermophilic bacterium Thermus aquaticus, which lives in hot springs, and it remains active (does not denature) during the high-temperature steps of PCR.
✓Final answer(a) Thermus aquaticus.
- CBSE 2026Set ANNUAL1 markMCQQ.Name the thermostable enzyme which is required during PCR and is isolated from bacterium Thermus aquaticus.(a) Taq - Polymerase(b) Pfu - Polymerase(c) DNA - Polymerase(d) DNA - Ligase
›Reveal solutionSolution
Taq Polymerase is a thermostable DNA polymerase from the thermophilic bacterium Thermus aquaticus, used in PCR because it survives the repeated high-temperature denaturation steps.
PCR (Polymerase Chain Reaction) requires repeated cycles of: (1) denaturation at ~94-95°C to separate the DNA strands, (2) annealing of primers at a lower temperature, and (3) extension/synthesis of new strands by a DNA polymerase. An ordinary DNA polymerase (like human/E. coli Pol I) would denature and lose activity at the high denaturation temperature every cycle. Taq polymerase, isolated from the hot-spring bacterium Thermus aquaticus, is naturally heat-stable and remains active through repeated heating cycles, making it ideal for PCR.
✓Final answer(a) Taq-Polymerase.
- CBSE 2025Set ANNUAL1 markMCQQ.The polymerase chain reaction is a technique used for(a) Amplification of DNA(b) Amplification of enzymes(c) Amplification of proteins(d) All of these
›Reveal solutionSolution
The Polymerase Chain Reaction (PCR) is an in-vitro technique to rapidly amplify (make many copies of) a chosen segment of DNA.
PCR uses a heat-stable DNA polymerase (commonly Taq polymerase, isolated from the thermophilic bacterium Thermus aquaticus), two short primers flanking the target DNA sequence, and repeated thermal cycles of denaturation (separating the DNA strands at high temperature), annealing (primers bind to the template), and extension (the polymerase synthesises new complementary strands). Each cycle roughly doubles the amount of the target DNA, so after ~20-30 cycles millions of copies of the specific DNA segment are produced. PCR is widely used in gene cloning, diagnostics, and forensic DNA fingerprinting.
✓Final answer(a) Amplification of DNA.
- CBSE 2025Set ANNUAL1 markQ.In PCR reaction at Step-2, denaturation of DNA takes place. Name the factor responsible for it.
›Reveal solutionSolution
The double-stranded template DNA is denatured by heating, which breaks the hydrogen bonds between the two strands.
Polymerase Chain Reaction (PCR) proceeds through repeated cycles of three steps. In the first step (denaturation), the reaction mixture is heated to a high temperature (typically 94-96°C), which breaks the hydrogen bonds holding the two DNA strands together, separating the double-stranded template into two single strands. This is followed by primer annealing at a lower temperature (~50-65°C) and primer extension by a thermostable DNA polymerase (Taq polymerase, from Thermus aquaticus) at ~72°C — the polymerase's heat-stability is what allows it to survive the repeated high-temperature denaturation steps.
✓Final answerHeat / high temperature.
- CBSE 2025Set ANNUAL1 markQ."Thermus aquaticus is preferred in PCR technique". Justify in one point.
›Reveal solutionSolution
Taq polymerase, from the thermophilic bacterium Thermus aquaticus, survives the repeated high-temperature denaturation steps of PCR, unlike ordinary DNA polymerases.
PCR (Polymerase Chain Reaction) involves repeated cycles of:
- Denaturation — heating the DNA to about 94–95°C to separate the two strands.
- Annealing — cooling to allow primers to bind to the template.
- Extension — using DNA polymerase to synthesise new complementary strands.
An ordinary DNA polymerase (such as from E. coli) would be denatured/destroyed at the high denaturation temperature and would need to be added fresh in every single cycle, which is impractical for the 20–30+ cycles used in PCR. Thermus aquaticus, however, is a bacterium that naturally lives in hot springs, and its DNA polymerase (Taq polymerase) is thermostable — it can withstand the repeated heating to ~95°C without losing its catalytic activity. This means the same enzyme added at the start of the reaction remains functional throughout all the PCR cycles, making the technique fast, simple, and automatable (as done in a thermal cycler).
✓Final answerThermus aquaticus is preferred in PCR because its DNA polymerase (Taq polymerase) is thermostable and remains active despite the repeated high-temperature (~95°C) denaturation steps, so it need not be added freshly in every cycle.
- CBSE 2025Set ANNUAL1 markQ.From the above diagram - Identify X and Y
›Reveal solutionSolution
In this PCR-cycle diagram, step (B) — where short primer sequences bind to the separated single DNA strands — is Annealing, and the structure labelled Y is the pair of Primers.
The diagram shows the three repeating steps of a PCR (Polymerase Chain Reaction) cycle: (A) Denaturation, (B) X, and (C) Extension.
- Step (A), Denaturation: the double-stranded DNA (ds DNA) is heated so the two strands separate into single strands.
- Step (B), labelled X: after denaturation, short primer sequences bind (anneal) to complementary sequences on each of the single DNA strands — this step is called Annealing.
- Step (C), Extension: Taq polymerase, using deoxynucleotides, extends the annealed primers to synthesise new complementary DNA strands.
The structure labelled Y in the diagram, shown binding to the single strands during the annealing step, refers to the Primers — the short oligonucleotide sequences that anneal to the template DNA strands.
✓Final answerX = Annealing (the step where primers bind to the single-stranded DNA); Y = Primers (the short DNA sequences that anneal to the template strands).
- CBSE 2025Set ANNUAL1 markQ.From the above diagram - Name the microbe from which a heat stable (thermostable) enzyme is isolated.
›Reveal solutionSolution
The heat-stable DNA polymerase used in PCR, called Taq polymerase, is isolated from the thermophilic bacterium Thermus aquaticus, which naturally lives in hot springs.
PCR requires the DNA polymerase to remain functional through repeated heating cycles (denaturation at ~94-95°C), which would destroy/denature an ordinary DNA polymerase. To overcome this, PCR uses a thermostable DNA polymerase called Taq polymerase, isolated from the bacterium Thermus aquaticus — a thermophilic (heat-loving) bacterium naturally found in hot springs, where it survives and functions at very high temperatures. Because this enzyme remains stable and active even after repeated exposure to the high denaturation temperatures of each PCR cycle, it does not need to be replenished after every cycle, which is what makes automated PCR possible.
✓Final answerThe heat-stable enzyme (Taq polymerase) used in PCR is isolated from the bacterium Thermus aquaticus.
- CBSE 2025Set ANNUAL1 markQ.From the above diagram - What is the role of Y ?
›Reveal solutionSolution
Primers (Y) are short DNA sequences that bind to the template strands and give Taq polymerase the free 3'-OH end it needs to start building the new strand.
Y refers to the primers used in PCR. Primers are short, chemically synthesised, single-stranded oligonucleotide sequences (typically about 18–24 bases long) that are complementary to the sequences flanking the region of DNA to be amplified.
Role of the primers: After the DNA is denatured into single strands, the primers anneal (bind by complementary base pairing) to their specific matching sequences on each of the single template DNA strands, one primer for each strand. DNA polymerase enzymes (like Taq polymerase) cannot initiate synthesis of a new DNA strand on their own — they can only extend an existing strand that already has a free 3'-hydroxyl (3'-OH) end. The bound primer supplies exactly this free 3'-OH end, giving Taq polymerase the starting point it needs to begin adding new nucleotides and synthesise the new complementary DNA strand during the extension step. Primers also determine specifically which segment of the DNA gets amplified, since only the region between the two primer-binding sites is copied.
✓Final answerThe primers (Y) anneal to the template DNA strands and provide the essential free 3'-OH starting point that Taq polymerase requires to begin synthesising the new DNA strand.
- CBSE 2025Set ANNUAL1 markQ.From the above diagram - What will happen to this biological process if X did not take place ?
›Reveal solutionSolution
Without annealing, the primers never attach to the template DNA, so DNA polymerase has no free 3'-OH end to extend from — no new DNA strand can be made, and PCR amplification does not occur.
X refers to the annealing step, in which the primers bind to their complementary sequences on the single-stranded template DNA. If this step did not take place:
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The primers would remain unbound/free in the reaction mixture, not attached anywhere on the single-stranded template DNA.
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Taq polymerase requires a primer already annealed to the template, providing a free 3'-OH end, in order to begin adding nucleotides and synthesising a new complementary strand — without an annealed primer, the polymerase has no site to begin extension.
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As a result, no new DNA strand synthesis (extension) could occur in that cycle.
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Since PCR relies on repeated cycles of denaturation, annealing, and extension to exponentially amplify the target DNA sequence, failure of the annealing step would mean the DNA could not be copied/amplified at all — the process would simply fail to produce any amplified product.
✓Final answerWithout annealing (X), the primers cannot bind the template DNA, so Taq polymerase has no starting point for synthesis, and PCR fails to amplify the DNA at all.
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- CBSE 2024Set 57/3/11 markMCQQ.Which one of the following represents the correct annealing of primers to the DNA to be amplified in the PCR ? (A) [diagram: two template strands each with a short primer annealed] (B) [diagram: two template strands each with a short primer annealed] (C) [diagram: two template strands each with a short primer annealed] (D) [diagram: two template strands each with a short primer annealed]
›Reveal solutionSolution
Primers must anneal to the 3' ends of each template strand in opposite orientations, with their own 3' ends pointing toward each other, to allow DNA polymerase to synthesize new strands in the 5' to 3' direction toward the target region.
The polymerase chain reaction depends entirely on the precise placement and orientation of primers. To understand which diagram is correct, you need to grasp what primers actually do and how DNA polymerase works.
DNA polymerase can only add nucleotides to the 3' end of an existing strand — it synthesizes in the 5' to 3' direction exclusively. This directional constraint shapes everything about primer design. When you want to amplify a specific segment of DNA, you need two primers: one that binds to the 3' end of the sense strand and another that binds to the 3' end of the antisense strand. Because the two strands of DNA run antiparallel (one 5' to 3', the other 3' to 5'), the primers must face each other from opposite ends of the target region.
Picture the target sequence you want to amplify sitting between two boundary points. The forward primer anneals to one template strand at one boundary, with its 3' end pointing inward toward the target. The reverse primer anneals to the complementary strand at the other boundary, also with its 3' end pointing inward. When polymerase extends both primers, the new strands grow toward each other, copying the entire region in between.
ImportantThe key recognition feature: both primers must have their 3' ends oriented toward the interior of the target sequence, not away from it. If a primer's 3' end points outward, polymerase will synthesize away from the target region, defeating the entire purpose of amplification.
A common error in diagrams shows primers annealed with their 3' ends pointing outward, or both primers on the same strand. Both configurations are biochemically useless. The first would amplify everything except your target; the second would leave one strand uncopied.
The correct diagram will show the two template strands separated (denatured), each with a short primer bound near opposite ends of the target region, and crucially, the 3' end of each primer must point toward the other primer. This arrangement ensures that when polymerase extends both primers simultaneously, the newly synthesized strands converge on and fully cover the target sequence.
✓Final answerThe correct primer annealing pattern shows one primer on each template strand, positioned at opposite ends of the target region, with both primers' 3' ends oriented inward toward each other so DNA polymerase synthesizes the complementary strands across the entire target sequence.
- CBSE 2024Set A11 markMCQQ.Which of the following statements are correct for the enzyme Taq polymerase? I. Taq polymerase is thermally unstable. II. It requires primers for carrying out the process of polymerisation. III. Taq polymerase is thermally stable. Choose the correct option.(a) I and II(b) I and III(c) II and III(d) I, II and III
›Reveal solutionSolution
Taq polymerase is thermally stable and requires primers, so statements II and III are correct.
Taq polymerase is a DNA polymerase isolated from the thermophilic bacterium Thermus aquaticus. It is used in PCR because it is thermally stable — it remains active and does not denature at the high temperatures (around 94-95 °C) used for denaturation. Like all DNA polymerases, it requires primers to initiate polymerisation. Therefore statement II (requires primers) and statement III (thermally stable) are correct, while statement I (thermally unstable) is wrong.
✓Final answer(c) II and III
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