Recombinant DNA are molecules formed by laboratory genetic recombination techniques 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.
Creation
Molecular cloning is a laboratory process used to create recombinant DNA. It is one of the two most widely used methods, along with the 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 is that molecular cloning involves replication in a living cell, while PCR involves in vitro. Another difference is that the first method allows cutting and pasting of DNA sequences, while the second is enhanced by copying the existing order.
Vector DNA
Getting recombinant DNA requires a cloning vector. It is derived from plasmids or viruses and is a relatively small segment. The choice of vector for molecular cloning depends on the choice of host organism, the size of the DNA to be cloned, and whether foreign molecules are to 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.
- Select host organism and cloning vector.
- Obtaining a DNA vector.
- Formation of cloned DNA.
- Creation of recombinant DNA.
- Introducing it into the host organism.
- Selection of organisms containing it.
- Selection of clones with desired DNA inserts and biological properties.
After transplantation into the host organism, the foreign molecules contained in the recombinant construct may or may not be expressed. Expression requires restructuring of the gene to include sequences that are necessary for DNA production. It is being used by the host's translation machine.
How it works
Recombinant DNA works when the host cell expresses a protein from recombinant genes. Expression depends on surrounding the gene with a set of signals that provide instructions for its transcription. They include promoter, ribosome binding and terminator.
Problems arise if the genecontains introns or signals that act as terminators for the bacterial host. This leads to premature termination. The recombinant protein may be improperly processed, folded, or degraded. Its production in eukaryotic systems usually occurs in yeasts and filamentous fungi. The use of animal cages is difficult due to the need for a strong supporting surface for many.
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 the polymerase chain reaction test.
In some cases, recombinant DNA can have harmful effects. This can happen when its fragment containing an active promoter is located next to a previously silent host cell gene.
Use
Recombinant DNA technology is widely used in biotechnology, medicine and research. Its proteins and other products can be found in almost every Western pharmacy, veterinary clinic, doctor's office, medical or biological laboratory.
The most common application is in basic research, where technology is essential to much of today's work in the biological and biomedical sciences. Recombinant DNA is used to identify, map and sequence genes, and to determine themfunctions. rDNA probes are used to analyze gene expression in single cells and in tissues of whole organisms. Recombinant proteins are used as reagents in laboratory experiments. Some specific examples are given below.
Recombinant chymosin
Found in abomasum, chymosin is an enzyme needed to make cheese. It was the first genetically modified food additive used in the industry. A microbiologically produced recombinant enzyme structurally identical to a calf-derived enzyme is cheaper and produced in larger quantities.
Recombinant human insulin
Virtually replaced insulin derived from animal sources (eg pigs and cattle) for the treatment of insulin-dependent diabetes. Recombinant insulin is synthesized by introducing the human insulin gene into bacteria of the genus Eterichia or yeast.
Growth Hormone
Prescribed for patients whose pituitary gland does not produce enough growth hormone to support normal development. Before recombinant growth hormone became available, it was obtained from the pituitary gland of cadavers. This unsafe practice has led some patients to develop Creutzfeldt-Jakob disease.
Recombinant coagulation factor
This is a blood-clotting protein that is administered to patients with forms of hemophilia with bleeding disorders. They are unable to producefactor VIII in sufficient quantities. Prior to the development of recombinant factor VIII, the protein was made by processing large amounts of human blood from multiple donors. This carried a very high risk of transmitting infectious diseases.
Diagnosis of HIV infection
Each of the three widely used methods for diagnosing HIV infection was developed using recombinant DNA. An antibody test uses her protein. It detects the presence of HIV genetic material using reverse transcription polymerase chain reaction. The development of the test was made possible by molecular cloning and sequencing of the HIV genomes.
