Vectors and Gene Preparation
To transfer foreign genetic material into a bacterium, specialized carrier molecules called vectors are required. Most commonly, these are bacteriophages or plasmids (extrachromosomal circular bacterial DNA molecules).
The process begins with the preparation of the vector and the donor DNA fragment containing the target gene. Both molecules are treated with the same restriction enzyme (restriction endonuclease). This step cuts the circular plasmid at a specific restriction site and yields a linear donor DNA fragment with identical complementary ("sticky") ends ready for joining.
Main Stages of Cloning
The creation and introduction of a recombinant molecule involve several sequential steps:
- Ligation. The previously prepared linearized plasmid and the foreign DNA fragment are joined together. This forms a chimeric plasmid (recombinant DNA) combining the vector's genetic material and the insert.
- Transformation. The chimeric molecule is introduced into a host cell (usually a bacterium). The bacterial cell now contains both its native chromosome (nucleoid) and the newly introduced plasmid.
- Selection (Screening). It is necessary to identify and isolate only those bacteria that have successfully taken up the recombinant DNA. Antibiotic resistance genes originally present in the plasmid are used for this purpose.
- Cultivation. The surviving, selected bacteria actively multiply on a nutrient medium. Cell division drives plasmid replication, effectively cloning the inserted DNA fragment.
Two Directions of Cell Utilization
After successful bacterial culture growth, subsequent steps diverge based on the experimental goal:
- Amplification (DNA production). If the researcher needs the cloned gene itself, recombinant plasmids are extracted from the accumulated bacterial biomass. They are treated again with the same restriction enzyme to excise the insert, and the target fragment is purified. The result is a massive yield of copies of the desired DNA molecule.
- Expression (Protein production). If the gene product is required, the bacterial culture is grown under specific conditions that stimulate transcription and translation of the cloned gene. Cells actively synthesize the target protein, after which they are lysed, and the protein product is purified.
Practical Applications of the Technology
Recombinant DNA technology revolutionized medicine, biology, and pharmacology. Key applications include:
- Microorganisms as protein producers. Bacteria synthesize vital human substances: protein hormones (insulin, growth hormone, somatostatin), biologically active peptides, vaccines (e.g., against hepatitis C), and clotting factors (factor VIII for hemophilia treatment).
- Gene therapy. Treatment of severe inherited disorders by introducing normal copies of defective genes into the body.
- Selective breeding. Creation of novel genetically modified plant and animal strains with targeted beneficial properties.