Cutting and joining DNA: restriction enzymes and ligase
Genetic modification starts with two jobs: cutting the gene you want out of one organism's DNA, and joining it into a piece of DNA that can be carried into another cell.
Restriction enzymes — the scissors
- A restriction enzyme cuts DNA at a specific sequence of bases (a specific site).
- Each restriction enzyme recognises one particular short base sequence, so it only cuts where that sequence occurs. This is why the cut is precise and the same gene is removed every time.
- Many restriction enzymes cut the two strands unevenly, leaving short lengths of unpaired bases at each end. These are called sticky ends.
- Sticky ends matter because their exposed bases will pair up (by complementary base pairing) with any other DNA cut by the same restriction enzyme.
Key exam sentence: "The same restriction enzyme is used to cut the human DNA and the plasmid, so both have complementary sticky ends."
Ligase — the glue
- Ligase joins pieces of DNA together by sealing the sugar–phosphate backbone.
- Once the sticky ends of the gene and the sticky ends of the plasmid have paired up, ligase joins them permanently.
- The result is a single piece of DNA made from two different sources — recombinant DNA.
The sequence in order
| Step | Enzyme | What happens |
|---|---|---|
| 1 | Restriction enzyme | Cuts the required gene out of the donor DNA, leaving sticky ends |
| 2 | Restriction enzyme (the same one) | Cuts open the plasmid, leaving matching sticky ends |
| 3 | — | Sticky ends pair up by complementary base pairing |
| 4 | Ligase | Joins the gene into the plasmid to form recombinant DNA |
Common mistake: writing "restrictive enzymes". The word is restriction. Marks are lost for the wrong technical term. Also do not write lipase — that digests lipids and has nothing to do with genetic modification.
Examiner tip. Spell the technical terms exactly: restriction enzyme (not 'restrictive enzyme') and ligase (not 'lipase'). Examiners repeatedly report candidates losing marks for these two errors, and for naming no enzyme at all — vague phrases like 'an enzyme cuts the DNA' score nothing.