Frequently tested concepts (2018–2024):
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EcoRI sequence and cut type (NEET 2019, 2021): Always 5'-GAATTC-3'; always produces sticky ends. Distractor: blunt ends. Never forget "staggered cut = sticky ends."
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Taq polymerase source (NEET 2018, 2022): Answer = Thermus aquaticus. Distractors: E. coli, Agrobacterium. Reason asked: thermostability at 94–98 °C.
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Gel electrophoresis migration direction (NEET 2020, 2023): DNA → anode (+). Distractor: cathode. Key reasoning: phosphate groups → negative charge → opposite charge attracts.
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PCR cycle sequence: Denaturation → Annealing → Extension. Temperatures: 94 °C → 50–65 °C → 72 °C. Know the formula: 2ⁿ copies after n cycles.
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pBR322 selectable markers (repeated annually): ampR and tetR — not chloramphenicol. Insertional inactivation = insert into tetR → loss of Tet resistance identifies recombinants.
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Ti plasmid and disarming (NEET 2021, 2023): Must be disarmed before use. T-DNA = transfer DNA. Agrobacterium primarily transforms dicots; gene gun used for monocots like rice and maize.
NEET question types for this chapter:
- Match the column (enzyme → recognition sequence / organism → vector)
- Assertion-Reason (e.g., "Taq polymerase is used in PCR because it is thermostable")
- Identify the incorrect statement (classic trap: checking pBR322 markers, PCR temperatures)
- Sequence-ordering questions (steps of DNA cloning or PCR cycle order)
Strategy:
- Learn exact temperatures and exact sequences — NEET uses these as distractors
- For match-the-column: link organism names to their contribution (Mullis = PCR, Smith/Nathans = restriction enzymes, Berg = recombinant DNA)
- When uncertain between anode and cathode, recall: "DNA is Negative → goes to Anode (Positive)"