Topic 21.3 · Supplement
The Process of Genetic Modification
Restriction enzymes and DNA ligase get mixed up constantly, and the easiest way to keep them apart is to remember what each one physically does: one cuts DNA, the other joins it. Everything in this sequence follows from that one distinction.
Cambridge uses the production of a human protein by bacteria as the worked example here, most often human insulin (see genetic modification: definition and examples). The process is a fixed, ordered sequence, and it’s worth learning it in that order rather than as a loose set of facts.
The sequence
- Identify and isolate the required gene — for example, the human insulin gene.
- A restriction enzyme cuts the DNA containing that gene, leaving sticky ends.
- The same restriction enzyme is used to cut a bacterial plasmid, producing matching, complementary sticky ends on the plasmid.
- The gene is inserted into the plasmid — the complementary sticky ends let the two pieces of DNA fit together.
- DNA ligase joins the DNA, producing a recombinant plasmid.
- The recombinant plasmid is inserted into a bacterial cell.
- The bacteria containing the recombinant plasmid are grown, and the inserted gene is expressed — the bacteria use the information in it to make the required protein.
Why using the same restriction enzyme matters
Cutting the human DNA and the plasmid with the samerestriction enzyme is what produces complementary sticky ends that can actually fit together — cut with two different enzymes, the ends wouldn’t match, and step four simply wouldn’t work. A sticky end is a short exposed section of DNA produced by that cut, and when two pieces of DNA have complementary sticky ends, their bases can pair up with each other.
Sticky ends only make the pieces fit — they don’t permanently join them into one continuous strand of DNA. That’s DNA ligase’s job specifically, and it’s the step that actually produces the recombinant plasmid: a plasmid containing DNA combined from more than one source. A useful way to keep the two enzymes apart under exam pressure is simply: restriction enzyme cuts DNA, DNA ligase joins DNA.
What happens after step six
Cambridge doesn’t require the specific details of how the recombinant plasmid actually gets into the bacterial cell — that detail sits outside the current syllabus depth. What matters is what happens once it’s in: as the bacteria reproduce, the population carrying the required gene increases, and the bacterium’s own machinery reads the inserted gene and builds the protein it codes for. This is exactly why bacteria make useful biological “factories” for human proteins, and those bacteria are then grown at industrial scale in a fermenter.
Important boundary
Advanced cloning methods and detailed plant-transformation protocols sit outside this process — the sequence above is specifically the bacterial gene-insertion process, not a general description of every genetic-modification technique that exists.