6.1.3(f)(i)-(ii) - Genetic engineering principles and techniques
Genetic engineering is the deliberate transfer of a gene from one organism into another organism so the recipient can gain a new genetic instruction. In this lesson, you will learn the method sequence: isolate a target gene, place it into a vector, form recombinant DNA, and introduce it into a host cell. The important skill is not memorising a story about one product; it is being able to explain why each tool is needed.
The Genetic Engineering Principle
The principle of genetic engineering is simple but powerful: a useful gene is isolated from one organism and placed into another organism using a suitable vector. The recipient organism then contains DNA that it did not previously have.
Genetic engineering
The deliberate manipulation of an organism's DNA, usually by transferring a gene from one organism into another organism.
The transferred gene may code for a useful protein such as insulin. A compatible promoter and regulatory sequences are needed for the host to express it; carrying a gene does not by itself guarantee protein production.
A vector is a carrier used to transfer DNA into a host cell. In this lesson, the key vector is a plasmid. A plasmid is a small circular DNA molecule, commonly found in bacteria, that can be used to carry a target gene into a host cell.
Recombinant DNA
DNA made by joining DNA from different sources, such as a bacterial plasmid joined to a gene from another organism.
The method has a logic:
| Step | Biological problem | Tool or idea that solves it |
|---|---|---|
| isolate the target gene | the useful sequence is part of a larger DNA molecule | restriction enzymes can cut DNA at specific sequences |
| place the gene into a vector | the gene needs a carrier into a host cell | a plasmid can act as a vector |
| make the DNA molecule stable | the gene and plasmid must be joined covalently | DNA ligase seals the DNA backbone |
| transfer into a host cell | DNA does not enter every cell by itself | electroporation increases membrane permeability |
In method answers, name the tool and its role. "Use enzymes" is too vague; "use a restriction enzyme to cut the plasmid and gene" is creditworthy.
Compatible DNA Ends
Restriction enzymes, also called restriction endonucleases, cut DNA at specific base sequences. These sequences are called recognition sites. In many simple genetic-engineering explanations, the same restriction enzyme is used to cut both the donor DNA containing the target gene and the plasmid vector.
Restriction enzyme
An enzyme that recognises a specific DNA base sequence and cuts DNA at or near that sequence.
Using the same restriction enzyme matters because it can produce matching cut ends on the gene and the plasmid. Many restriction enzymes make staggered cuts, leaving short single-stranded overhangs called sticky ends. Sticky ends with complementary bases can pair by hydrogen bonding.
Think of the sequence like this:
- The target gene is cut out of donor DNA.
- The plasmid vector is cut open.
- The cut gene and cut plasmid have compatible ends.
- The gene can line up with the opened plasmid.
A vector should have a suitable single cutting site outside essential regions, including its origin of replication. Multiple cuts can fragment the plasmid; cutting an essential control region can prevent its maintenance.
The restriction enzyme does not "stick" the gene into the plasmid. It cuts DNA. Complementary sticky ends help the fragments align, but another enzyme is needed to form the permanent covalent joins.
| Incorrect shortcut | Mechanistic explanation |
|---|---|
| "The enzyme inserts the gene" | "A restriction enzyme cuts the target gene and cuts open the plasmid." |
| "The plasmid chooses the gene" | "The plasmid is cut to produce ends compatible with the target gene." |
| "Sticky ends are strong enough on their own" | "Sticky ends can anneal, then DNA ligase seals the sugar-phosphate backbone." |
Making Recombinant DNA
Once the target gene and plasmid vector have compatible ends, the DNA fragments can anneal. Annealing means complementary single-stranded regions pair with each other by hydrogen bonds. That pairing is useful, but it is not enough to make a stable recombinant plasmid.
DNA ligase is the enzyme that joins the DNA fragments permanently. It catalyses the formation of phosphodiester bonds in the sugar-phosphate backbone. The product is recombinant DNA because the plasmid now contains DNA from a different source.
DNA ligase
An enzyme that joins DNA fragments by forming phosphodiester bonds in the sugar-phosphate backbone.
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