Explain why a restriction endonuclease and DNA ligase are both required when inserting a human gene into a bacterial plasmid vector.

OCR A-Level Biology A (H420) — 6.4 Cloning and biotechnology · Explain · 4 marks · View as Markdown

Written & reviewed by James Millett — Biology (Imperial College London), PGCE Science (University of Cambridge).

Genetic engineers routinely transfer genes from human cells into bacterial plasmids so that bacteria can be used to manufacture human proteins such as insulin on an industrial scale.

Model answer (4 marks)

A restriction endonuclease recognises a specific DNA sequence and cleaves the phosphodiester bond, producing two DNA fragments with complementary sticky ends. The same enzyme is used to cut the plasmid vector, giving it compatible over‑hanging ends. The human gene fragment, cut with the same enzyme, has matching sticky ends, so the two pieces can anneal through hydrogen bonding of the complementary single‑stranded overhangs. DNA ligase then catalyses the formation of a phosphodiester bond between the adjacent 5′‑phosphate and 3′‑hydroxyl groups, sealing the sugar‑phosphate backbone and creating a stable recombinant plasmid.

Examiner tips

  • Use the word ‘restriction endonuclease’ and explain it cuts at a specific sequence; mention sticky ends. Show that the same enzyme gives compatible ends on plasmid and insert. Explain annealing via complementary overhangs. State that DNA ligase joins the backbone to stabilise the recombinant plasmid.

Common mistakes

  • Failing to mention that the same enzyme gives compatible sticky ends on both pieces. Confusing blunt ends with sticky ends. Omitting the role of DNA ligase in sealing the backbone.

Mark scheme (4 marks)

  1. Restriction endonuclease cuts the DNA at specific recognition sequences
  2. This produces sticky ends (complementary/unpaired single-stranded overhangs) on both the plasmid and the human gene fragment
  3. Complementary sticky ends allow the human gene and plasmid to anneal/base-pair by hydrogen bonding
  4. DNA ligase seals/joins the sugar-phosphate backbone, forming a stable recombinant plasmid

Key terms in this question

restriction endonuclease · plasmid · DNA ligase

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