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

The technique of bringing about improvements in living organisms by genetic modifications and hybridization, for the welfare of human beings is known as ‘Biotechnology’.

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.

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.

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

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

• Biotechnology, a term coined by Karl Ereky in 1919, is the use of biological systems and genetic modifications to develop products and services for human welfare.
• Traditional biotechnology relies on small-scale, natural processes like fermentation (e.g., producing curd and wine), whereas modern biotechnology operates on a large scale.
• Modern biotechnology is fundamentally driven by two core techniques: genetic engineering (the targeted alteration of DNA and RNA) and bioprocess engineering.
• The field experienced a major breakthrough with the development of recombinant DNA technology by Cohen and Boyer in 1973.
• By integrating disciplines such as molecular biology and biochemistry, biotechnology enables crucial applications in both medicine (antibiotics, vaccines, insulin) and agriculture (high-yield, disease-resistant crops).

• Two Core Techniques – Modern biotechnology is based on (i) genetic engineering and (ii) chemical engineering.
• Genetic Engineering – Deals with the alteration of DNA and RNA to achieve desired results in a directed, predetermined way using in vitro processes.
• Chemical Engineering - Maintains a sterile environment for manufacturing useful products like vaccines, antibodies, enzymes, vitamins, and therapeutics.
• What Genetic Engineering Involves - Repairing/replacing defective genes, synthesising new genes, transferring genes, combining genes from two organisms, and altering the genotype.
• Other Names for Genetic Engineering – Also called Recombinant DNA (rDNA) Technology or Gene Cloning, as it involves transferring a gene via a suitable vector to a new location or organism.

• Genetic engineering, also known as recombinant DNA technology, involves manipulating and transferring genes from one organism to another to create a new DNA structure.
• This technology is highly effective because the fundamental genetic code is similar across all living species, allowing genes to be successfully shared and expressed.
• Scientists utilise these techniques to study how genes function, understand how life processes are regulated, and clone specific DNA fragments.
• In medicine, it has allowed for the mass production of vital compounds, such as producing human insulin using bacteria or human growth hormone in cow's milk.
• In agriculture, it is used to create genetically modified foods that possess highly desirable traits, such as improved nutritional value and enhanced resistance to pests and diseases.

• Recombinant DNA technology manipulates and joins DNA from different sources using three main tools: enzymes, cloning vectors, and competent hosts.
• Restriction enzymes act as "molecular scissors" to cut DNA at specific sites, while DNA ligase acts as "glue" to seal the fragments together.
• Cloning vectors (such as plasmids or bacteriophages) serve as vehicles for carrying, inserting, and replicating foreign DNA within a host cell.
• Competent hosts are cells that have been specially treated (using techniques such as heat shock or gene guns) so they can successfully take up the foreign recombinant DNA.

• Restriction enzymes, often called "molecular scissors", are specialised endonucleases that cut DNA at specific internal positions to facilitate genetic engineering.
• Over 900 restriction enzymes, including the first discovered Hind II, have been successfully isolated from more than 230 strains of bacteria.
• These enzymes are named using a standard convention based on the genus, species, strain, and discovery order of the source bacterium, such as EcoRI.
• They function by inspecting DNA to find specific palindromic nucleotide sequences, which are base pair sequences that read the same on both strands in the same orientation.
• Type II restriction enzymes are the primary ones used in genetic manipulation because they reliably recognise and cut at very specific target sequences.
• Cutting the DNA slightly away from the centre of a palindrome creates "sticky ends", which are single-stranded overhangs that easily bond with complementary DNA using the enzyme DNA ligase.
• The resulting DNA fragments are separated by size using gel electrophoresis, where smaller negatively charged fragments move faster through an agarose matrix toward an anode.
• The separated DNA is visualised as bright orange bands under UV light after ethidium bromide staining and is carefully extracted from the gel through a process called elution.

• An ideal cloning vector must contain an origin of replication to initiate and control the multiplication of the inserted foreign DNA.
• It requires a selectable marker, such as antibiotic resistance genes, to distinguish and isolate successfully transformed cells from non-transformed ones.
• The vector should ideally possess a single recognition site for common restriction enzymes to easily link the foreign DNA without complicating the cloning process.
• Insertional inactivation techniques, such as blue-white selection or the loss of antibiotic resistance, are utilised to clearly identify recombinant colonies.
• The vector must be small in size to ensure easy manipulation and to prevent the DNA from breaking down during laboratory purification procedures.

• Plasmids are small, circular, double-stranded DNA molecules that replicate independently of the host bacterial chromosome and naturally confer antibiotic resistance.
• Due to their manageable size and precisely mapped DNA sequences, plasmids serve as highly effective and easily isolatable vectors for genetic cloning.
• A typical plasmid vector includes critical functional regions such as an origin of replication, recognition sites for endonucleases, and antibiotic resistance genes for selection.
• Recombinant DNA is created by cutting both the target foreign DNA and the plasmid with specific restriction enzymes and joining them together using DNA ligase.
• This newly formed recombinant plasmid is then inserted into a host bacterium, which multiplies rapidly to produce large quantities of the desired foreign gene.

• Agrobacterium tumefaciens is a bacterial pathogen that specifically infects several types of dicot plants.
• The bacterium possesses a specialised plasmid, widely known as the Ti plasmid, which contains a transferable DNA segment called T-DNA.
• During an infection, the pathogen successfully inserts a piece of this T-DNA directly into the DNA of the host plant.
• This genetic insertion alters normal cellular functions, transforming healthy plant cells into tumour cells.
• Consequently, the inserted T-DNA directs these newly formed tumour cells to synthesise and produce specific chemicals required by the pathogen.

• Phages typically possess linear DNA molecules that allow for the insertion of foreign DNA at various specific restriction enzyme sites.
• Chimeric or recombinant DNA is successfully collected after the phage completes its lytic cycle and produces mature, infective particles.
• Plasmids and viruses serve as vectors, acting as biological carriers to effectively transport foreign DNA into target organisms.
• By interchanging plasmid and viral DNA fragments, scientists synthesise new vectors capable of delivering genes into diverse bacterial, yeast, insect, plant, and animal cells.
• The biological process of successfully introducing these foreign DNA fragments into a suitable host cell is formally known as transformation.

• The tumour-inducing (Ti) plasmid of Agrobacterium tumefaciens and retroviruses serve as highly effective vectors for cloning genes into plant and animal hosts, respectively.
• Once a desired DNA fragment is successfully ligated into a suitable vector, it is transferred into a host organism to undergo multiplication.
• A gene library is a comprehensive collection of cloned DNA fragments that completely represents the entire genome of an organism.
• Genomic libraries are prepared from total DNA and contain both introns and exons, whereas cDNA libraries represent mRNA populations and contain only exons.

• Cloning is the process of producing an exact genetic copy of a gene, cell, tissue, or organism.
• Gene cloning isolates and multiplies a single DNA segment to produce medically useful proteins.
• Reproductive cloning fuses a somatic cell nucleus with an enucleated egg to produce a complete organism.
• Therapeutic cloning derives stem cells from cloned embryos for disease treatment and medical research.
• Dolly the sheep was the first mammal cloned from an adult somatic cell using somatic cell nuclear transfer (SCNT).
• Cloning is associated with low technical efficiency, health abnormalities in offspring, and significant ethical concerns.

• Cells are first broken open using specific enzymes (such as lysozyme for bacteria) to successfully isolate the genetic material.
• The purified DNA is precisely cut at specific locations using restriction enzymes, which act as "molecular scissors" to extract the desired gene.
• The resulting DNA fragments are separated by size using gel electrophoresis, and the specific target sequence is extracted.
• The desired gene is then amplified into millions of copies using the polymerase chain reaction (PCR) technique.
• The amplified gene is joined to a carrier vector using the enzyme DNA ligase to construct a new molecule called recombinant DNA.
• This recombinant DNA is introduced into a chemically treated, competent host cell (such as a bacterium) through a process known as transformation.
• For commercial use, these transformed host cells are cultured on a massive scale inside large, environmentally controlled vessels called bioreactors.
• The final therapeutic product undergoes downstream processing, which involves rigorous separation, purification, and quality testing before packaging.

• Genetic engineering facilitates the study of gene structures and allows for the mass production of vital medical therapeutics, such as human insulin.
• Gene therapy directly treats inherited genetic disorders by replacing or complementing defective genes with functional ones.
• Genetically modified plants and animals are developed to enhance agricultural efficiency, improve nutritional value, and potentially reduce reliance on chemical fertilisers.
• Industrial-scale manufacturing of these biological products requires bioreactors, which provide strictly controlled, optimal environments for large cell cultures.
• The widespread use of genetic modification raises significant ethical, ecological, and health concerns that require careful ongoing evaluation.

• Accessing an individual's genetic information regarding behaviours or predispositions to diseases can lead to severe ethical violations of privacy and human rights.
• While genetic screening enables beneficial prenatal diagnosis and medical treatments, it simultaneously poses risks of social discrimination in areas such as employment and health insurance.
• It is crucial for governments to establish strict legal frameworks and regulatory bodies, such as India's GEAC, to carefully oversee genetic research and protect public safety.
• The prospect of human cloning raises profound moral dilemmas, as the technology could theoretically be exploited to intentionally replicate highly specific human traits or personalities.
• The creation of cloned human beings introduces unprecedented social complexities, particularly concerning the ethical challenges and family dynamics of raising a child without traditional biological parents.

• Genetic transformation in animals is achieved by introducing foreign DNA into fertilised eggs or embryos.
• Microinjection method involves direct injection of foreign DNA into the pronucleus of a fertilised 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.