Skip to main content
SingaporeBiotechnologySyllabus dot point

How do scientists cut DNA at exactly the right place and then glue pieces back together?

Describe the action of restriction enzymes and DNA ligase and explain how they are used to make recombinant DNA

A focused answer to the O-Level outcome on cutting and joining DNA. Restriction enzymes and recognition sites, sticky ends, the role of DNA ligase, and making recombinant DNA.

Generated by Claude Opus 4.89 min answer

Reviewed by: AI editorial process; not yet individually human-reviewed

Have a quick question? Jump to the Q&A page

Jump to a section
  1. What this dot point is asking
  2. The answer
  3. Examples in context
  4. Try this

What this dot point is asking

This outcome asks you to describe what restriction enzymes and DNA ligase do, and how they work together to make recombinant DNA. These two enzymes are the molecular scissors and glue of genetic engineering, and understanding them unlocks almost every technique that follows.

The answer

Restriction enzymes: the molecular scissors

A restriction enzyme is an enzyme that cuts DNA at a specific base sequence called its recognition site.

  • Each restriction enzyme recognises one particular sequence and cuts only there, so it is highly specific.
  • Many restriction enzymes make a staggered cut, leaving short single-stranded overhangs called sticky ends.
  • Because the cut is at a fixed sequence, the same enzyme always produces the same fragments and the same sticky ends.

Sticky ends

Sticky ends are useful because they are single-stranded and can pair, by complementary base pairing, with any other sticky end made by the same enzyme. This is what lets pieces of DNA from different sources join together.

DNA ligase: the molecular glue

DNA ligase is an enzyme that joins pieces of DNA by sealing the sugar-phosphate backbone. After two sticky ends have paired up, ligase forms the bonds that make the join permanent.

Making recombinant DNA

Putting the two enzymes together:

  1. Cut the gene from its source with a restriction enzyme, leaving sticky ends.
  2. Cut the plasmid (the vector) with the same restriction enzyme, giving complementary sticky ends.
  3. Mix them so the sticky ends pair by base pairing.
  4. Add DNA ligase to seal the backbone, forming a recombinant plasmid containing the gene.

The result, DNA combining sequences from two sources, is called recombinant DNA.

Examples in context

Example 1. Inserting the insulin gene. The human insulin gene and a bacterial plasmid are cut with the same restriction enzyme, their sticky ends pair, and ligase seals them. The recombinant plasmid then directs the bacteria to make insulin, the foundation of recombinant protein production.

Example 2. A defence system turned into a tool. Restriction enzymes evolved in bacteria to chop up invading viral DNA. Scientists borrowed this natural defence and now use it to cut DNA precisely, a neat example of nature's tools being repurposed for biotechnology.

Try this

Q1. State what a restriction enzyme does to DNA. [1 mark]

  • Cue. It cuts DNA at a specific recognition sequence, often leaving sticky ends.

Q2. Explain why the gene and the plasmid must be cut with the same restriction enzyme. [2 marks]

  • Cue. So they have complementary sticky ends that can pair by base pairing, allowing them to be joined.

Q3. State the role of DNA ligase in making recombinant DNA. [1 mark]

  • Cue. It seals the sugar-phosphate backbone, joining the paired DNA pieces permanently.

Exam-style practice questions

Practice questions written in the style of SEAB exam questions on this dot point, with worked answer explainers. The year tag is the paper they imitate, not the source.

Original6 marksExplain how restriction enzymes and DNA ligase are used together to insert a gene into a plasmid to form recombinant DNA.
Show worked answer →

Examiners want the two enzymes' roles and the reason for using the same restriction enzyme.

A restriction enzyme cuts DNA at a specific recognition sequence. When it cuts, it often leaves short single-stranded overhangs called sticky ends. The gene to be inserted and the plasmid are cut with the same restriction enzyme, so they have complementary sticky ends.

When the cut gene and plasmid are mixed, the complementary sticky ends pair up by base pairing. DNA ligase then joins them by sealing the sugar-phosphate backbone, producing recombinant DNA, a plasmid that now carries the inserted gene.

What markers reward: restriction enzyme cutting at a specific sequence leaving sticky ends, using the same enzyme on gene and plasmid for complementary ends, base pairing of the sticky ends, and DNA ligase sealing the backbone to form recombinant DNA.

Original3 marksExplain why a restriction enzyme is described as specific, and why this specificity is useful.
Show worked answer →

The answer should link specificity to the recognition sequence and its usefulness.

A restriction enzyme is specific because it recognises and cuts only at one particular base sequence, its recognition site, and not at other sequences. This is like a lock that fits only one key.

This specificity is useful because it lets scientists cut DNA at chosen, predictable places, so the same enzyme always produces the same fragments and the same sticky ends, allowing pieces to be joined reliably.

What markers reward: the idea that the enzyme cuts only at a specific recognition sequence, and that this allows DNA to be cut at predictable, chosen places giving consistent, matching ends.

Related dot points