Revision: Biotechnology and Its Applications >> Principles and Processes of Biotechnology Biology (Theory) ISC (Science) ISC Class 12 CISCE

The European Federation of Biotechnology (EFB) defined biotechnology as ‘the integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services.’

Genetic engineering is the manipulation and transfer of genes from one organism to another organism to create a new DNA called recombinant DNA (rDNA). Genetic engineering is also called recombinant DNA technology.

Plasmids are small, circular, double-stranded DNA molecules found in bacteria that replicate independently of chromosomal DNA and often carry antibiotic-resistance genes.

Phages are viruses that infect bacteria and contain linear DNA into which foreign DNA fragments can be inserted for cloning purposes.

A gene library is a collection of cloned DNA fragments that together represent the complete genome of an organism.

Gene cloning is a genetic engineering technique in which a single copy of a gene or DNA segment is isolated and multiplied to produce many identical copies.

The process of producing an exact genetic replica of a cell, tissue, organ, or entire organism is called cloning.

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Cloning is the process of producing identical copies of a gene, DNA fragment, cell, or organism.

The production of a complete organism by fusion of a somatic cell nucleus with an enucleated ovum is called reproductive cloning.

The technique of producing stem cells from cloned embryos for treatment of diseases is called therapeutic cloning.

• Recombinant DNA technology depends on the isolation, cutting, and joining of DNA fragments to form chimeric DNA.
• Restriction enzymes act as molecular scissors that cut DNA at specific sequences to obtain desired genes.
• DNA ligase joins DNA fragments by forming covalent bonds, producing recombinant DNA molecules.
• Cloning vectors are DNA molecules that carry foreign DNA into host cells for replication.
• Common vectors include plasmids, bacteriophages, cosmids, and phasmids, which replicate independently inside host cells.

• Origin of replication (ori): Enables the vector and inserted DNA to replicate inside the host cell and controls copy number.
• Selectable marker: Marker genes (e.g., antibiotic resistance) help identify and select transformed and recombinant cells.
• Cloning sites: Presence of unique restriction enzyme sites allows insertion of foreign DNA without fragmenting the vector.
• Small size: Small vectors are easy to isolate, handle, and manipulate during cloning.

1. Isolation of DNA:
   Genetic material (DNA) is extracted and purified from the source organism by removing proteins, RNA, lipids, and other impurities.
2. Cutting and Separation of DNA:
   DNA is cut at specific sites using restriction endonucleases, and desired fragments are separated by gel electrophoresis and isolated by elution.
3. Amplification of Gene (PCR):
   The gene of interest is amplified using Polymerase Chain Reaction (PCR) to produce millions of copies for effective cloning.
4. Ligation and Gene Transfer:
   The desired DNA fragment is ligated into a vector and introduced into a host cell by methods such as transformation, electroporation, or gene gun.
5. Expression and Production:
   The host cells expressing the recombinant gene are cultured on a large scale in bioreactors to produce the desired recombinant protein.
6. Downstream Processing:
   The final product is separated, purified, and processed to obtain a market-ready product.

1. Understanding gene structure and expression:
   Genetic engineering helps in studying gene structure, regulation, exons, introns, and mechanisms of gene expression.
2. Medical applications:
   Used to produce therapeutic proteins such as insulin, growth hormone, and enzymes, and to diagnose and predict genetic diseases.
3. Industrial applications:
   Enables large-scale production of hormones, medicines, enzymes, and industrial chemicals through recombinant microorganisms.
4. Agricultural applications:
   Development of genetically modified crops with higher yield, better nutrition, pest resistance, and reduced dependence on fertilizers.
5. Gene therapy:
   Used to treat genetic disorders by replacing defective genes, such as in SCID and experimental treatments in animals and humans.
6. Research and future potential:
   Genetic engineering has vast potential but also raises concerns about environmental, health, and ethical risks, requiring careful regulation.

• Genetic transformation in animals is achieved by introducing foreign DNA into fertilized eggs or embryos.
• Microinjection method involves direct injection of foreign DNA into the pronucleus of a fertilized egg and is widely used to produce transgenic animals.
• Nuclear replacement transfers a somatic cell nucleus into an enucleated egg to produce genetically identical offspring.
• Cloning allows multiplication of desirable transformed genomes to produce transgenic animals.