The trap first: Students confuse the role of PCR with the role of blotting. PCR amplifies — it makes millions of copies of a specific DNA segment. Blotting detects — it separates molecules, transfers them to a membrane, and visualises a specific target using a probe. They are different stages of molecular analysis, and NEET questions routinely test whether you can distinguish "amplify" from "detect."
PCR (Polymerase Chain Reaction): Invented by Kary Mullis. Uses a thermostable DNA polymerase (Taq polymerase, from Thermus aquaticus) to amplify a target DNA sequence in vitro. Each cycle has three steps — denaturation (~95°C), annealing (~55–65°C), and extension (~72°C). Two primers are essential: a forward primer and a reverse primer, each flanking the target region on opposite strands, oriented inward. After n cycles, the target is amplified approximately 2ⁿ times (NCERT Class 12 Biology Chapter 9, pages 172–173).
Gel electrophoresis: Separates DNA fragments by size through an agarose matrix under an electric field. Smaller fragments migrate faster toward the anode. Visualised using ethidium bromide staining under UV.
Blotting techniques:
Watch-out: "Which technique amplifies DNA?" — the answer is always PCR, never a blotting method. Blotting identifies what is already present; PCR generates copies.
Select an option to see the explanation. Wrong answers show why your choice was tempting — and name the exact trap it exploits.
The enzyme used in PCR is isolated from a thermophilic bacterium because it must withstand:
Answer: D. Taq polymerase is thermostable — it survives repeated denaturation at ~95°C without losing activity, which is the defining requirement for PCR cycling (NCERT Class 12 Biology Chapter 9, page 173).
Why A is wrong: A is wrong because PCR does not involve pH changes between steps; the buffer remains constant. This confuses PCR conditions with electrophoresis buffer chemistry.
Why B is wrong: B is wrong because UV is used post-electrophoresis for visualisation with ethidium bromide, not during PCR amplification itself (trap: PCR vs blot role confusion).
Why C is wrong: C is wrong because extension occurs at ~72°C (elevated, not low temperature). This reverses the thermal profile of PCR.
PCR requires two primers because:
Answer: A. Two primers — forward and reverse — anneal to opposite strands at the boundaries of the target sequence, pointing inward. Together they bracket the amplicon so that both strands are copied (NCERT Class 12 Biology Chapter 9, pages 166–167).
Why B is wrong: B is wrong because primers do not correspond to PCR steps; both function during the annealing step. Denaturation uses heat, not a primer (mistake: confusing primer role with cycle step).
Why C is wrong: C is wrong because if both bound the same strand, only one strand would be copied and exponential amplification would not occur. Primers must flank opposite strands.
Why D is wrong: D is wrong because both PCR primers are short synthetic DNA oligonucleotides, not RNA. RNA primers are used in in vivo replication, not in PCR.
After 10 cycles of PCR starting from a single double-stranded DNA molecule, the approximate number of copies of the target region is:
Answer: B. PCR amplification is exponential: 2ⁿ copies after n cycles. 2¹⁰ = 1024 (NCERT Class 12 Biology Chapter 9, pages 172–173).
Why A is wrong: A is wrong because 20 = 2×10, which treats amplification as linear (additive) rather than exponential. PCR doubles the target each cycle.
Why C is wrong: C is wrong because 512 = 2⁹, which is one cycle short. Students sometimes start counting from cycle 0 or miscalculate the exponent.
Why D is wrong: D is wrong because 100 = 10² confuses the exponent relationship. The base is 2 (doubling), not 10.
In gel electrophoresis of DNA fragments, smaller fragments migrate:
Answer: B. DNA is negatively charged (phosphate backbone) and migrates toward the positive electrode (anode). Smaller fragments encounter less resistance in the gel matrix and therefore travel farther/faster (NCERT Class 12 Biology Chapter 9, page 168).
Why A is wrong: A is wrong because DNA moves toward the anode (positive), not cathode (negative). DNA carries a net negative charge.
Why C is wrong: C is wrong because while the direction (anode) is correct, smaller fragments move faster, not slower. Larger fragments are retarded more by the gel pores.
Why D is wrong: D is wrong on both counts — DNA migrates toward the anode, and smaller fragments move faster, not slower.
Southern blotting is used to detect:
Answer: D. Southern blotting: DNA fragments separated by gel electrophoresis are transferred to a membrane and hybridised with a complementary labelled DNA/RNA probe to detect a specific sequence. Named after Edwin Southern (NCERT Class 12 Biology Chapter 9, page 168).
Why A is wrong: A is wrong because RNA detection is Northern blotting, not Southern. The naming convention: Southern = DNA, Northern = RNA (trap: PCR vs blot role confusion — and blot-type confusion).
Why B is wrong: B is wrong because protein detection using antibodies is Western blotting, not Southern.
Why C is wrong: C is wrong because Southern blotting specifically requires a labelled probe for hybridisation-based detection. Without a probe, you have only gel separation, not blotting.
A researcher wants to determine whether a patient carries a specific mutant allele. She amplifies the region using PCR, then separates the products by gel electrophoresis. The technique she uses to confirm the identity of the band is:
Answer: C. PCR amplifies DNA non-specifically with respect to confirming sequence identity — size alone may not distinguish alleles. Southern blotting with a sequence-specific probe confirms the identity of a DNA fragment by hybridisation (NCERT Class 12 Biology Chapter 9, page 168).
Why A is wrong: A is wrong because PCR amplifies a region defined by primer binding sites but does not itself confirm internal sequence identity. A band of the correct size could still be a non-specific product (trap: PCR vs blot — PCR amplifies, blot detects).
Why B is wrong: B is wrong because Western blotting detects proteins, not DNA sequences. The sample here is amplified DNA.
Why D is wrong: D is wrong because Northern blotting detects RNA, not DNA. The researcher is working with a PCR-amplified DNA product.
During PCR, the step in which primers bind to their complementary sequences on the template DNA is called:
Answer: C. Annealing (typically 55–65°C) is the step where primers hydrogen-bond to their complementary sequences on the single-stranded template after denaturation separates the strands (NCERT Class 12 Biology Chapter 9, page 173).
Why A is wrong: A is wrong because denaturation (~95°C) separates the double-stranded DNA into single strands. Primers cannot bind during this step — it's too hot.
Why B is wrong: B is wrong because extension (~72°C) is when Taq polymerase synthesises new DNA from the primer's 3' end. Binding has already occurred during annealing.
Why D is wrong: D is wrong because ligation (joining fragments with ligase) is a cloning step, not a PCR step. PCR does not use ligase.
A student claims that PCR uses only one primer per reaction because "polymerase extends in both directions from a single primer." The error in this reasoning is:
Answer: A. DNA polymerase synthesises exclusively in the 5'→3' direction. To amplify both strands of a double-stranded target, you need two primers: one on each strand, oriented inward. A single primer would only copy one strand linearly (mistake: saying PCR uses one primer — it uses two).
Why B is wrong: B is wrong because the premise is false — DNA polymerase does NOT extend in both directions. It only extends 5'→3'. The error in the student's claim is more fundamental than just exponential kinetics.
Why C is wrong: C is wrong because DNA polymerase cannot initiate synthesis de novo; it absolutely requires a primer with a free 3'-OH. Removing primers would halt PCR entirely.
Why D is wrong: D is wrong because primers are not consumed by heat during denaturation. They are small oligonucleotides that re-anneal each cycle. The need for two primers relates to strand directionality, not thermal degradation.
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Given
A forensic scientist has a blood sample containing trace DNA from a suspect. She needs to determine whether the suspect's DNA contains a specific 500 bp sequence linked to a genetic marker.
Available tools: Taq polymerase, primers flanking the 500 bp region, agarose gel apparatus, Southern blot kit with a probe complementary to the 500 bp marker.
Required
Identify the correct sequence of techniques and their roles in confirming the presence of the specific 500 bp marker.
Concept
PCR amplifies a specific DNA region (defined by primers) to detectable quantities. Gel electrophoresis separates the amplified product by size. Southern blotting confirms sequence identity by probe hybridisation — because a 500 bp band on a gel could be any 500 bp fragment; only probe binding proves it is the target.
Formula/Rule
• PCR: 2ⁿ amplification (exponential, n = number of cycles)• Gel: migration ∝ 1/log(size) — smaller fragments travel farther• Southern blot: sequence-specific hybridisation (complementary probe binds target)
Substitution/Application
1. PCR the trace DNA using the two primers → amplify the 500 bp target region.
2. Gel electrophoresis → run the PCR product; look for a band at ~500 bp.
3. Southern blot → transfer gel to membrane → hybridise with the labelled probe → confirm the 500 bp band IS the marker (not a non-specific product of similar size).
Calculation/Reasoning
If PCR runs for 30 cycles: 2³⁰ ≈ 1.07 × 10⁹ copies of the target — sufficient for gel visualisation and blotting. The number 30 is an exact counting integer (does not limit significant figures).
Final answer
Correct technique sequence: PCR → Gel electrophoresis → Southern blotting. PCR amplifies, gel separates by size, Southern confirms identity. Each technique has a distinct, non-interchangeable role.
Common trap
Confusing amplification (PCR) with detection (blotting). A common wrong answer would say "Southern blotting amplifies DNA" or "PCR confirms identity." Remember: Amplify = PCR. Detect specific sequence = blot.
Similar NEET-style question
"Which of the following correctly pairs the technique with its primary function?
(a) PCR — sequence-specific detection
(b) Southern blot — DNA amplification
(c) Northern blot — RNA detection
(d) Western blot — DNA transfer to membrane"
Answer: (c). Northern blot detects specific RNA by hybridisation.
---
PCR: denature 94°C → anneal 50-65°C → extend 72°C with Taq pol. Gel electrophoresis: agarose (DNA) or PAGE (proteins) — separation by size in electric field. Southern (DNA), Northern (RNA), Western (protein) blots.
-- NCERT Class 12 Biology, Chapter 9, p. 168These are the exact patterns that cause wrong answers in NEET. Each trap includes when it triggers and how to avoid it.
Category: Similar Terms
PCR amplifies (makes copies); blotting transfers + visualises specific bands. Different stages of analysis.
Question asks which technique amplifies vs detects.
Amplify = PCR. Detect specific = blot (Southern DNA, Northern RNA, Western protein).
Root cause: concept gap
PCR requires TWO primers — one for each end of target DNA, anneal to opposite strands, point inward. Together they bracket the amplicon.
More in Biotechnology and its Applications: 5 exam traps and mistakes · 1 question pattern from its other lessons.
9 questions from NEET 2020, 2022, 2023, 2024, 2025, 2026. Answers verified against NTA official keys.
During PCR, primers bind to the DNA strands in the ______ step.
Polymerase chain reaction (PCR) amplifies DNA following the equation.
Which of the following statements is incorrect?
Upon exposure to UV radiation, DNA stained with ethidium bromide will show
Which one of the following statement is not true regarding gel electrophoresis technique?
Transposons can be used during which one of the following ?
In gel electrophoresis, separated DNA fragments can be visualized with the help of :
All 40 past-paper questions from Biotechnology and its Applications →
Page numbers are the ones printed in the current NCERT textbook (2023 rationalised edition), unless marked pre-2023. The books are free at ncert.nic.in.
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