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).

Scientists use recombinant DNA technology to introduce human genes into bacterial cells so that the bacteria produce useful proteins, such as insulin, on an industrial scale. A key step involves cutting and joining DNA from two different organisms.

Model answer (4 marks)

A restriction endonuclease recognises a specific DNA sequence and cuts both the plasmid and the human gene, producing complementary sticky ends. The sticky ends allow the human gene to anneal to the plasmid by hydrogen bonding. DNA ligase then seals the sugar‑phosphate backbone, forming a stable covalently bonded recombinant plasmid.

Examiner tips

Mention the specific role of the endonuclease (recognition and cutting) and that the same enzyme gives complementary sticky ends.
Explain that sticky ends enable base‑pairing between the insert and vector.
State that ligase seals the nicks to create a continuous backbone.
Use the exact terms ‘restriction endonuclease’, ‘sticky ends’, ‘anneal’, ‘DNA ligase’, ‘covalently bonded’.

Common mistakes

Confusing restriction enzymes with ligase or vice versa.
Failing to mention that the same enzyme gives complementary ends on both DNA fragments.
Omitting the step where ligase seals the backbone.

Mark scheme (4 marks)

Restriction endonuclease cuts the DNA at a specific recognition sequence
Cutting with the same restriction endonuclease produces complementary sticky ends on both the plasmid and the human gene
Sticky ends allow the human gene to anneal/hydrogen bond to the open plasmid
DNA ligase seals the sugar-phosphate backbone to form a stable, covalently bonded recombinant plasmid

Key terms in this question

restriction endonuclease · plasmid · vector · DNA ligase