How It Works: Restriction Enzymes, Ligase, and Vectors

How It Works: Restriction Enzymes, Ligase, and Vectors

Genetic engineering relies on precise molecular tools to cut, carry, and insert genes. Here is the detailed mechanism:

1. Identify the gene: Scientists locate the gene that codes for the desired protein — for example, the human insulin gene found on chromosome 11.
2. Cut with restriction enzymes: Restriction enzymes act as molecular scissors. Each restriction enzyme recognises a specific short DNA sequence (restriction site) and cuts through the double strand at that point, leaving "sticky ends" — short single-stranded overhangs that allow complementary DNA to join on.
3. Prepare the vector: A plasmid (a small circular ring of bacterial DNA used as a vector) is cut open with the same restriction enzyme, giving it the same sticky ends.
4. Insert the gene: The gene with sticky ends is mixed with the open plasmid. Complementary base pairing causes the sticky ends to join. DNA ligase enzyme then permanently seals (ligates) the joins, creating a recombinant plasmid containing the human gene.
5. Transform the host organism: The recombinant plasmid is transferred into bacteria (e.g., Escherichia coli). The bacteria take up the plasmid — this process is called transformation.
6. Expression and harvest: The bacteria transcribe and translate the inserted gene, producing the desired protein. Bacteria are cultured in large fermenters; the protein is extracted and purified. For insulin production, this replaced the older method of extracting insulin from pig or cow pancreases.