Genetic Engineering is a branch of biotechnology in which the manipulation of DNA occurs to obtain a desired product, also known as Recombinant DNA technology or Genetic modification. It allowed scientists to add, remove, and modify genetic materials, mainly to change the phenotypic traits.
The steps of Genetic Engineering are shown below:
1. Identification of the target gene
- To identify the segment of a particular gene that we want to add, remove, and modify in an organism.
- Genomic sequencing, Hybridisation techniques, Mutational studies, Protein analysis, and Gene databases are some of the methods that are used to identify the target gene.
- Example: for Insulin production, the target gene is the human insulin gene.
2. Isolation of the Gene
- Isolation of the gene is the process by which the desired gene is separated from the DNA of the organism. That gene carries the particular information of that trait.
- PCR (Polymerase Chain Reaction), Agarose gel electrophoresis, and cDNA methods, which are used in gene isolation.
- Restriction endonucleases cut DNA segments at specific locations.
- DNA ligase is used to join DNA fragments.
- Example: The Insulin gene is isolated from pancreatic mRNA through restriction enzymes or synthesised via cDNA.
3. Selection of a Suitable Vector
- A vector acts as a vehicle that carries the desired gene and transfers it into the host organism safely.
- Origin of replication (Ori), Selectable marker genes, Restriction sites, Small size, Non-toxic are the characteristics of an ideal vector.
- Six types of vectors are used in genetic engineering, as listed below:
Type | Role |
|---|---|
Plasmids | small, double-stranded DNA that can replicate independently. |
Viral vectors | engineered viruses that transfer foreign DNA into a host organism. |
Bacteriophages | It is a virus that affects and multiplies inside bacteria. |
Cosmids | It is an artificial plasmid-bacteriophage vector to clone a large DNA segment. |
BACs (Bacterial Artificial Chromosomes) | Genetically engineered vector to clone large DNA fragments in bacteria, especially in E.coli. |
YACs (Yeast Artificial Chromosomes) | To clone huge DNA fragments |
4. Insertion of Gene into Vector
- Formation of Recombinant DNA.
- Cutting the vector DNA using the Restriction Endonuclease enzyme, cutting the target gene with the same Restriction endonuclease enzyme, and then joining the fragments by using DNA ligase are the three steps of Gene insertion.
5. Transfer of Recombinant DNA into Host Cell
- To transfer Recombinant DNA into a host organism where it replicates and expresses the desired gene.
- It is done by three methods: Transformation, Transfection, and Transduction, depending on the method and host type.
- Transformation- In this process, the Recombinant DNA is introduced into the bacterial cells.
- Transfection- In this process, the introduction of rDNA into a Eukaryotic cell happens.
- Transduction - In this process, the Bacteriophage transfers the rDNA from one bacterium to another.
6. Screening and Selection of Transformed Cells
- To identify and separate transformed cells from non-transformed cells.
- In bacteria, the Antibiotic resistance marker method is used, in which transformed cells survive on antibiotic media.
- In blue- white screening method, colony colour differentiates between recombinant and non-recombinant cells. In which blue colour colony means non-recombinant cells are present, and a white colony means recombinant cells are present.
7. Expression of the Gene in Host Organism
- To ensure the desired gene is expressed in the host organism and produces the desired proteins or traits.
- Bacteria (E. coli), plant cells, animal cells, yeast, and mammals are the host systems which is used in gene expression.
- RT-PCR (checks mRNA level), Western Blot (Confirms protein production), ELISA (measures physical quantities), and Reporter genes (Visual confirmation) are the methods in which how measure gene expression is measured.
8. Extraction and Purification of Gene Product
- To extract and purify the gene products such as insulin, vaccines, enzymes, and hormones.
- Chromatography is the most common method that is based on the separation of size, charge, or affinity.
- It ensures that the product is biologically active, removes toxins, and is critical for biopharmaceutical quality and safety.
Importance of Genetic Engineering
- Production of therapeutic proteins such as recombinant insulin, clotting factors, and growth hormone.
- To detect early diagnosis through genetic probes and PCR.
- Production of enzymes, antibodies, vitamins, and amino acids.
- Production of Biofortified crops like Golden Rice that have high vitamin A to reduce malnutrition.
- Manufacture of biofuels and bioplastics.
- Development of transgenic model organisms for disease research, like rats.