The Process of Genetic Modification
Plasmids: These are small, circular loops of extra-chromosomal DNA found in bacteria. They are ideal vectors because they are easy to isolate, manipulate in a test tube, and re-insert into bacterial cells.
Viruses: Viruses can also act as vectors by 'infecting' a target cell with the recombinant DNA. This method is often used when the target is a human cell or a specific type of bacteria that does not easily take up plasmids.
Function of a Vector: The vector's primary role is to carry the foreign gene into the host cell and ensure that the gene is replicated and expressed by the host's cellular machinery.
Isolation: The specific gene required for the trait (e.g., the human insulin gene) is identified and located within the donor's genome.
Cutting: Restriction enzymes are used to cut the gene out of the donor DNA. Crucially, the same restriction enzyme is used to cut the vector DNA (the plasmid), ensuring both have matching, complementary sticky ends.
Ligation: The isolated gene and the opened plasmid are mixed together. DNA ligase is added to chemically bond the fragments, creating a recombinant plasmid.
Transformation: The recombinant plasmid is inserted into a host cell, such as a bacterium. This is often achieved through chemical or electrical treatments that make the cell membrane more permeable.
Expression & Cloning: The host bacteria are grown in large volumes (often in a fermenter). As they reproduce, they copy the recombinant plasmid and begin to synthesize the protein encoded by the inserted gene.
| Feature | Restriction Enzymes | DNA Ligase |
|---|---|---|
| Function | Cuts DNA at specific sequences | Joins DNA fragments together |
| Analogy | Molecular Scissors | Molecular Glue |
| Result | Creates sticky ends | Creates a continuous DNA strand |
The 'Same Enzyme' Rule: Always emphasize that the same restriction enzyme must be used for both the gene and the vector. If different enzymes are used, the sticky ends will not be complementary, and the DNA will not join.
Terminology Precision: Use the term 'recombinant' to describe the DNA or the organism once the foreign gene has been successfully integrated.
Why Bacteria?: If asked why bacteria are used, remember three points: they share the same universal genetic code, they contain easily manipulated plasmids, and there are fewer ethical concerns compared to using animals.
Common Sequence: Be prepared to describe the process in order: Isolate Cut Join Insert Replicate.