Recombinant DNA is a molecule formed by laboratory methods of genetic recombination to combine genetic material from multiple sources. It is possible because the DNA molecules of all organisms have the same chemical structure and differ only in the nucleotide sequence within it.
Creature
Molecular cloning is a laboratory process used to create recombinant DNA. This is one of the two most widely used methods, along with polymerase chain reaction (PCR). It allows you to control the replication of any particular DNA sequence chosen by the experimenter.
There are two fundamental differences between recombinant DNA methods. One of them is that molecular cloning involves replication in a living cell, and PCR in vitro. Another difference is that the first method allows cutting and pasting DNA sequences, and the second is enhanced by copying the existing sequence.
DNA vector
The preparation of recombinant DNA requires a cloning vector. It comes from plasmids or viruses and is a relatively small segment. The choice of vector for molecular cloning depends on the choice of the host organism, the size of the cloned DNA, and whether foreign molecules should be expressed. Segments can be combined using various methods, such as restriction enzyme / ligase cloning or Gibson assembly.
Cloning
In standard protocols, cloning involves seven steps.
- The choice of the host organism and the cloning vector.
- Obtaining a DNA vector.
- The formation of cloned DNA.
- The creation of recombinant DNA.
- Its introduction into the host organism.
- The selection of organisms containing it.
- Selection of clones with desired DNA inserts and biological properties.
After transplantation into the host organism, foreign molecules contained in the recombinant construct can be expressed or non-expressed. Expression requires gene restructuring to include sequences that are necessary for DNA production. It is used by the host's broadcast apparatus.
How does it work
Recombinant DNA works when a host cell expresses a protein from recombinant genes. Expression depends on the environment of the gene with a set of signals that provide instructions for its transcription. They include a promoter, ribosome binding, and a terminator.
Problems arise if the gene contains introns or signals that act as terminators for the bacterial host. This leads to premature termination. Recombinant protein may be improperly processed, folded, or degraded. Its production in eukaryotic systems usually occurs in yeast and filamentous fungi. The use of animal cells is difficult due to the fact that many people need a strong supporting surface.
The properties of organisms
Organisms containing recombinant DNA molecules have apparently normal phenotypes. Their appearance, behavior and metabolism usually do not change. The only way to demonstrate the presence of recombinant sequences is to examine the DNA itself using a polymerase chain reaction test.
In some cases, recombinant DNA can be harmful. This can happen when its fragment containing the active promoter is located next to the previously silent gene of the host cell.
Using
Recombinant DNA technology is widely used in biotechnology, medicine, and research. Its proteins and other products can be found in almost every western pharmacy, veterinarian, doctor’s office, medical or biological laboratory.
The most common application is basic research, in which technology is important for most modern work in the biological and biomedical sciences. Recombinant DNA is used to identify, map and sequence genes, as well as to determine their function. RDNA probes are used to analyze gene expression in individual cells and in the tissues of whole organisms. Recombinant proteins are used as reagents in laboratory experiments. Some specific examples are given below.
Recombinant Chymosin
Found in the abomasum, chymosin is an enzyme necessary for the production of cheese. It was the first genetically modified food supplement used in industry. A microbiologically produced recombinant enzyme structurally identical to that obtained from a calf is cheaper and is produced in large quantities.
Recombinant Human Insulin
Almost completely replaced insulin obtained from animal sources (e.g. pigs and cattle) for the treatment of insulin-dependent diabetes. Recombinant insulin is synthesized by introducing a gene of human insulin into bacteria of the genus esterichia or yeast.
A growth hormone
It is prescribed for patients in whom the pituitary gland does not generate enough growth hormone to maintain normal development. Before recombinant growth hormone became available, it was obtained from the pituitary gland of corpses. This unsafe practice has led some patients to develop Creutzfeldt-Jakob disease.
Recombinant Coagulation Factor
This is a blood coagulation protein that is administered to patients with hemophilia with a bleeding disorder. They are not able to produce factor VIII in sufficient quantities. Prior to the development of recombinant factor VIII, a protein was obtained by treating large amounts of human blood from several donors. This carried a very high risk of transmission of infectious diseases.
HIV diagnosis
Each of the three commonly used methods for diagnosing HIV infection has been developed using recombinant DNA. An antibody test uses its protein. It determines the presence of HIV genetic material using reverse transcription polymerase chain reaction. Test development was made possible by molecular cloning and sequence analysis of HIV genomes.