Describe the method of recombinant plasmids. What steps can be distinguished in the creation and cloning of recombinant plasmids? What is the significance of this method?
This method includes:
1) Restriction – cutting a DNA molecule, for example, a mammalian cell, by enzymes-rextractases and into fragments with the same sticky ends and the desired gene. Plasmid DNA is cut with the same enzymes; therefore, the sticky ends of the plasmid are complementary to the nucleotide sequences of the sticky ends of the gene. A gene can also be synthesized artificially using matrix reactions. This synthesis is carried out using the enzyme reverse transcriptase, or revertase.
2) Ligation – by “stitching / Sticky ends into plasmid DNA using ligase enzymes and obtaining a recombinant plasmid.
3) Transformation – the introduction of a recombinant plasmid into a bacterial cell. To do this, the cell is exposed to high temperature and calcium chloride, which makes its membrane permeable to DNA. The recombinant ilazmide introduced into the bacterial cell begins to work, and the cell synthesizes a foreign protein. The frequency of plasmid entry into the cell is low (in one cell out of a thousand). The recombinant plasmid in the bacterial cell doubles many times, and the foreign gene multiplies, it is cloned, that is, the transfer from the mother cell to the daughter when; asexual reproduction. A colony is formed from each bacterial cell, consisting of millions of bacteria that are screened.
4) Screening – selection of bacterial colonies containing recombinant plasmids with the desired gene. For this, all colonies are covered with a special filter to which they adhere. Then the filter is treated with a radioactive probe – a polynucleotide containing in its composition a radioactive isotope of phosphorus – 32P.The radioactive probe is complementary to the desired gene, therefore it only binds to those bacterial colonies that have recombinant plasmids. To detect them, an X-ray film is applied to the filter, which is then developed. According to the position of the areas illuminated on the film, those colonies that received the desired gene are selected.
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