Vectors and gene preparation
To transfer foreign genetic material into a bacterium, special carrier molecules are required—vectors. Most commonly, these are bacteriophages or plasmids (bacterial extrachromosomal circular 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 enzyme—a restriction endonuclease. This is necessary to cleave the circular plasmid at a specific restriction site and obtain a linear donor DNA fragment with identical complementary ("sticky") ends ready for joining.
Main stages of cloning
The procedure for creating and introducing a recombinant molecule includes several strict sequential steps:
- Ligation. The previously prepared linearized plasmid and the foreign DNA fragment are joined together. As a result, a chimeric plasmid (recombinant DNA) is formed, combining the vector's genetic material and the insert.
- Transformation. The chimeric molecule is introduced into the host cell (bacterium). Now the bacterial cell contains not only its own chromosome (nucleoid), but also the newly introduced plasmid.
- Selection (screening). It is necessary to identify and select only those bacteria that have successfully taken up the recombinant DNA. For this, antibiotic resistance genes initially contained in the plasmid are used.
- Cultivation. The surviving selected bacteria actively multiply on a nutrient medium. During cell division, plasmid replication occurs, which means direct cloning of the inserted DNA fragment.
Two directions of cell utilization
After successful cultivation of the bacterial culture, further actions branch into two directions depending on the final goal of the experiment:
- Amplification (obtaining DNA). If the researcher needs the cloned gene itself, recombinant plasmids are isolated from the accumulated bacterial biomass. Then they are treated again with the same restriction enzyme to excise the insert, and the target fragment is purified. The result is the production of a huge number of copies of the desired DNA molecule.
- Expression (obtaining protein). If the gene product is needed, the bacterial culture is grown under specific conditions that stimulate transcription and translation of the cloned gene. Cells actively produce the target protein, after which they are lysed, and the finished protein product is thoroughly purified.
Practical application of the technology
The mechanism of using recombinant DNA has revolutionized medicine, biology, and pharmacology. The main areas of application include:
- Using microorganisms as producers. Bacteria synthesize substances vital to humans: protein hormones (insulin, growth hormone, somatostatin), biologically active peptides, vaccines (e.g., against hepatitis C), and blood clotting factors (factor VIII for the treatment of hemophilia).
- Gene therapy. Treatment of severe hereditary diseases by introducing normal copies of defective genes into the organism.
- Selection. Creation of fundamentally new genetic forms of plants and animals with specified useful properties.
Briefly: answers to topical questions
Which enzyme performs the joining of the plasmid and the foreign DNA fragment at the ligation stage?
The joining of the plasmid and the foreign DNA fragment is performed by DNA ligase. This enzyme covalently joins the fragments by restoring phosphodiester bonds between adjacent nucleotides.
As a result of this process, the integrity of the sugar-phosphate backbone of the molecule is restored, and recombinant (hybrid) DNA is formed. The following methods of fragment joining are distinguished:
- Via "sticky" ends — through the formation of hydrogen bonds between single-stranded regions of complementary nucleotides.
- Using artificially built "sticky" ends.
- Via "blunt" ends.
What types of restriction enzymes are used in genetic engineering?
In genetic engineering, about 200 restriction enzymes (restriction endonucleases) are used, which are classified according to the type of DNA molecule cleavage.
| Type of cleavage | Mechanism | Result |
|---|---|---|
| Along the axis of symmetry | Cleavage along the axis of symmetry of the recognition sequence | Double-stranded "blunt" ends are formed |
| With a shift | Cleavage with a shift, resulting in a "step" | "Sticky" ends with mutually complementary regions are formed |
"Sticky" ends can be formed at the 5'-end (phosphate) or the 3'-end (hydroxyl).
How to memorize the basic cloning sequence: RLTSC (Restriction — Ligation — Transformation — Selection — Cultivation). «Restrict the linear — target specific copies».
Class and exam questions
Why is it necessary to treat the plasmid and donor DNA with the same restriction enzyme?
This is necessary to create identical "sticky ends". Only in this case will the donor DNA fragment be able to ideally integrate into the cut plasmid according to the principle of complementarity.
How to separate bacteria with a chimeric plasmid from cells without it?
Through antibiotic selection. The plasmid carries a drug resistance gene. When grown on a medium with this antibiotic, only those bacteria that have successfully taken up the vector survive.
What is the difference between amplification and expression of a cloned gene?
Amplification aims to obtain multiple copies of the DNA molecule itself. Expression, however, implies that the bacterium "reads" the introduced gene and synthesizes the target protein based on it.
What else to study on this topic
- Mechanism of action of restriction endonucleases
- DNA ligase enzyme and molecular basics of ligation
- Methods of bacterial cell transformation
- Principles of gene expression induction in bacteria
- Methods of cell lysis and recombinant protein purification
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