Applications of Recombinant DNA Technology Chapter Notes | Biotechnology for Class 12 - NEET PDF Download

DNA Fingerprinting

• Recombinant DNA (rDNA) technology has revolutionized various aspects of life, including establishing individual identity, introducing foreign genes, diagnosing diseases, and producing therapeutic agents.
• DNA fingerprinting, developed by Sir Alec Jeffreys in 1984 at the University of Leicester, identifies inherited variations in human DNA without sequencing.
• Human DNA consists of four nitrogenous bases: Adenine (A), Guanine (G), Cytosine (C), and Thymine (T), with a specific arrangement unique to each individual.
• The human genome contains 3.2 × 10^9 base pairs, with 99.9% similarity among individuals and 0.1% consisting of inherited variations that make each person unique.
• More than 90% of the human genome is non-coding DNA, containing short nucleotide sequences repeated in tandem, known as Variable Number Tandem Repeats (VNTRs).
• VNTRs are subdivided into microsatellites (1–9 bp repeated sequences) and minisatellites (10–100 bp repeated sequences).
• VNTRs exhibit high polymorphism, forming the basis of DNA fingerprinting, and are inheritable, making them useful for paternity testing.
• DNA fingerprinting uses Restriction Fragment Length Polymorphism (RFLP), where DNA is digested with restriction enzymes to produce unique fragment patterns.
• In practice, identifying individual bands via RFLP is challenging, so Southern Hybridisation with VNTR probes is used to generate characteristic band patterns.
• Steps of DNA fingerprinting include:
  • Isolating DNA from samples like blood, hair, skin, semen, or buccal swabs.
  • Cutting DNA into fragments of varying sizes using restriction enzymes.
  • Separating DNA fragments by agarose gel electrophoresis based on size.
  • Transferring separated DNA to a nitrocellulose or nylon membrane and fixing it using UV radiation or baking at 80°C.
  • Performing Southern Hybridisation with labeled VNTR probes to detect complementary sequences.
  • Detecting hybridized DNA fragments to produce individual-specific band patterns.
• Polymerase Chain Reaction (PCR) is often used to amplify DNA, enhancing the sensitivity of DNA fingerprinting regardless of sample size.
• Applications of DNA fingerprinting include:
  • Ascertaining paternity and maternity using inherited VNTR patterns, allowing reconstruction of parental patterns from offspring.
  • Analyzing DNA from crime scenes (e.g., blood, hair, semen) to compare VNTR patterns with suspects, aiding in criminal identification and forensic studies.
  • Comparing DNA from fossils to modern counterparts, contributing to evolutionary biology studies.
  • Identifying DNA patterns associated with inherited disorders by comparing profiles of affected and unaffected individuals.
  • Proposing VNTR patterns as genetic barcodes for personal identification, alongside social security numbers or picture IDs.

Transgenic Organism

• Transgenic organisms, or genetically modified organisms (GMOs), carry a foreign gene (transgene) inserted into their genome through transgenesis, which is transmitted and expressed in their progeny.
• Examples include Bt Cotton (a transgenic plant) and Rosie the cow (a transgenic animal), created for human benefit and not naturally occurring.
• Historical milestones in GMO development:
  • 1973: Herbert Boyer and Stanley Cohen created the first GMO, a bacterium.
  • 1974: Rudolf Jaenisch and Beatrice Mintz engineered the first transgenic animal (mice).
  • 1994: Flavr Savr tomato became the first GM food crop approved by the USFDA.
  • 1975: Asilomar conference established guidelines for genetic engineering experiments.
  • 1982: FDA approved Humulin, the first pharmaceutical produced using genetic engineering.
  • 1995: US EPA approved the first insecticide-producing crop and Golden Rice to address Vitamin A deficiency.
  • 2009: FDA approved ATryn, the first biological product from a GM animal.
• Transgenic plants are produced by modifying their genome through genetic engineering, introducing genes from other species using vector-mediated (indirect) or vector-less (direct) gene transfer methods.
• Vector-mediated gene transfer methods:
  • Bacteria-mediated transfer using Agrobacterium tumefaciens, a Gram-negative soil bacterium that naturally transfers DNA (T-DNA) into plant genomes, causing crown gall disease.
  • Agrobacterium’s Ti plasmid contains T-DNA, which encodes phytohormones (auxin, cytokinins) inducing tumor formation.
  • Tumor-inducing genes are replaced with genes of interest (e.g., Bt gene for insect resistance) in a disarmed Ti plasmid, which is introduced into Agrobacterium for plant transformation.
  • Antibiotic marker genes differentiate transformed from non-transformed cells.
  • Transformed plant cells are cultured on regeneration media with bacteriostatic agents and antibiotics to eliminate non-transformed cells, forming shoots and roots for soil transfer.
  • Plant virus-mediated transfer using caulimoviruses, though less common, introduces DNA into plant cells.
• Vector-less gene transfer methods:
  • Particle bombardment (biolistics): DNA with genes of interest and antibiotic markers is coated onto gold or tungsten particles (1–3 µm) and bombarded onto target tissues using a gene gun.
  • Bombarded tissues are cultured on antibiotic-containing media to select transformed cells, which regenerate into transgenic plants.
  • This method is effective for cereals and chloroplast transformation, enabling high transgene expression due to multiple chloroplast copies.
  • Protoplast transformation and electroporation: Protoplasts (naked plant cells without cell walls) uptake DNA, accelerated by polyethylene glycol (PEG), dextran sulphate, or electric currents (electroporation).
  • Transformed protoplasts regenerate cell walls, divide, and are selected on selective media to produce transgenic plants.
• Transgenic animals are created using:
  • DNA pronuclear microinjection: Transgenes are injected into the male pronucleus of fertilized eggs, cultured in vitro, and implanted into foster mothers. Integration is random and success rates are low.
  • Embryonic stem cell-mediated gene transfer: Transgenes are introduced into embryonic stem (ES) cells via electroporation, which are then injected into blastocysts and implanted into foster mothers for offspring screening.
  • Retrovirus-mediated gene transfer: Retroviral vectors (e.g., lentivirus) introduce a single transgene copy into early embryos or ES cells, integrating at specific genomic locations.
• Chimeric mice, composed of genetically distinct cell lineages, are used to study developmental processes, cell differentiation, and disease mechanisms.
• Applications of transgenic plants:
  • In agriculture: Enhance crop yield and nutritional quality to meet population demands.
  • Develop resistance to biotic stresses (pathogens, insects) and abiotic stresses (drought, salinity, extreme temperatures).
  • Bt cotton, expressing Bt toxin from Bacillus thuringiensis, resists insects like pink bollworm, commercially grown in India since 2002.
  • Other Bt crops include brinjal, maize, rice, and tomato, targeting specific pests (e.g., cryIAc for cotton bollworms, cryIAb for corn borers).
  • Traits like herbicide tolerance, bacterial/viral resistance, nutrient enhancement (e.g., Golden Rice with vitamin A), and delayed fruit ripening improve productivity.
  • Antisense technology silences gene expression using antisense oligonucleotides (ASO) or RNA interference (RNAi) to produce pest-resistant plants (e.g., nematode-resistant tobacco).
  • In industry (Molecular Farming): Plants produce vaccine antigens, therapeutic proteins (e.g., Elelyso for Gaucher’s disease in carrot cells), diagnostic reagents, bioplastics, and enzymes.
  • For environmental benefits: Produce biodegradable plastics (e.g., polyhydroxybutyrate in sugarcane) and perform phytoremediation (e.g., mercury detoxification using mercuric reductase genes).
• Applications of transgenic animals:
  • In industry (Molecular Pharming): Produce recombinant proteins in milk, e.g., Tracy the ewe (α1-antitrypsin) and Rosie the cow (α-lactalbumin-enriched milk).
  • Other proteins include albumin (cow), growth hormone (goat), and coagulation factors (mouse, rabbit).
  • In research: Model organisms for studying human physiology, development, and diseases (e.g., cancer, Alzheimer’s).
  • Used for toxicity testing of vaccines, drugs, and chemicals.
• Concerns over GMOs include potential health risks (e.g., allergenicity), environmental impacts (e.g., invasiveness), and unpredictable effects from random transgene insertion.
• Viral vectors and promoters in GMOs may cause infections, raising safety concerns.
• In India, the Genetic Engineering Appraisal Committee (GEAC) under the Ministry of Environment, Forest and Climate Change regulates GMO manufacture, use, import, export, and storage.

Gene Therapy

• Gene therapy repairs faulty genes by introducing correct genetic material into cells to treat diseases caused by absent or defective genes.
• Candidate diseases include Severe Combined Immunodeficiency (SCID), cystic fibrosis, hemophilia, and muscular dystrophy.
• The first gene therapy in 1990 treated a four-year-old girl with SCID due to a defective adenosine deaminase (ADA) gene, improving immune function temporarily.
• Approaches for gene therapy:
  • Gene replacement/addition: A functional gene replaces a defective one, e.g., replacing mutated CFTR gene in cystic fibrosis or p53 in cancer cells.
  • Gene inhibition: Inactivates disease-causing genes using antisense RNA or other techniques, applicable to diseases like Tay-Sachs, phenylketonuria, and color-blindness.
  • Gene repair/editing: Modifies defective genes using CRISPR/Cas9, adapted from bacterial defense systems, to snip and replace mutated DNA.
• Types of gene therapy:
  • Ex vivo: Cells are removed, modified with normal genes in culture, and reintroduced into the patient (cell-based delivery).
  • In vivo: Normal genes are directly introduced into target cells/tissues (direct delivery).
• Merits include potential cures for inherited diseases and cancer through genetic manipulation.
• Demerits include uncontrolled therapeutic gene expression, random integration risks (e.g., tumor formation), short-lived effects, immune rejection, high costs, and ineffectiveness for multigene disorders.
• Ethical concerns:
  • Gene enhancement for traits like intelligence or athletic ability requires strict regulation.
  • Germline alterations, though potentially curative, may cause long-term side effects.
  • Gene therapy’s promise often exceeds its current delivery, but it is expected to improve for single-gene defects.

Recombinant Vaccines

• Vaccines are preparations of killed/weakened pathogens or their components that elicit immune responses to confer protection against diseases.
• The term “vaccine” was coined by Edward Jenner, derived from “vacca” (Latin for cow), who used cowpox to protect against smallpox.
• Vaccination, coined by Louis Pasteur, involves injecting pathogens or antigens to induce immunity, eliciting rapid responses upon re-exposure.
• Common vaccines include DPT (diphtheria, pertussis, tetanus) and MMR (measles, mumps, rubella).
• Conventional vaccines risk infection, toxoid reversal, or contamination, prompting the development of recombinant vaccines using rDNA technology.
• Types of recombinant vaccines:
  • Live genetically modified vaccines: Pathogens are attenuated by deleting/inactivating genes, or vector-based vaccines carry foreign genes (e.g., Salmonella, Pseudorabies vaccines).
  • Vaccinia virus, a poxvirus, is used to create polyvalent vaccines by inserting antigenic genes from multiple pathogens (e.g., hepatitis, herpes, influenza).
  • Recombinant subunit vaccines: Contain pathogen components (peptides/proteins) expressed in prokaryotic (E. coli) or eukaryotic (yeast) systems, e.g., Hepatitis B vaccine using HBsAg expressed in Saccharomyces cerevisiae.
  • DNA vaccines: Antigenic genes in plasmids are injected into muscles (via gene gun or nasal spray), inducing humoral and cellular immunity.
  • RNA vaccines: mRNA encoding antigens is injected, expressed in cells, and protected by lipid nanoparticles (LNPs) for stability.
• Edible plant vaccines, produced in plants like bananas, stimulate mucosal immunity upon consumption (e.g., Hepatitis B vaccine in bananas).
• RNA vaccines offer rapid development, high-yield production, and safety, with applications in COVID-19 (e.g., mRNA vaccines in trials).

Therapeutic Agents/Molecules: Monoclonal Antibodies, Insulin and Growth Hormone

• rDNA technology enables large-scale production of safe, pure human proteins, including antibiotics, monoclonal antibodies, blood clotting factors, hormones, cytokines, and vaccines.
• Monoclonal antibodies (MAbs) are specific to a single antigen epitope, unlike polyclonal antibodies, and are used in diagnostics and therapeutics.
• Hybridoma technology (Kohler and Milstein, 1975) fuses B cells with immortal myeloma cells using polyethylene glycol (PEG) to produce MAbs.
• Myeloma cells are engineered to die in HAT medium, while B cells survive briefly; only hybridoma cells survive long-term, producing MAbs.
• rDNA technology produces chimeric or humanized antibodies with human segments for higher efficacy.
• MAb applications include diagnostic kits (e.g., pregnancy tests), cancer imaging, and targeted therapies with attached toxins/drugs.
• Insulin, a hormone from pancreatic beta cells, regulates glucose uptake and glycogenesis; its deficiency causes diabetes mellitus.
• Insulin consists of A (21 amino acids) and B (30 amino acids) chains linked by disulfide bonds, derived from preproinsulin (110 amino acids) via proinsulin (86 amino acids).
• Animal-derived insulin (porcine, bovine) caused allergic reactions due to minor amino acid differences.
• In the 1970s, rDNA technology produced human insulin by cloning A and B chain genes in E. coli, fused with β-galactosidase, and cleaved with cyanogen bromide to form Humulin (1982).
• Human growth hormone (HGH), a 191-amino-acid peptide from the pituitary, promotes growth and metabolism; its deficiency causes dwarfism.
• Conventional HGH from cadavers risked Creutzfeldt-Jakob disease, prompting rDNA production.
• HGH gene synthesis removed the signal peptide (26 amino acids) using EcoRI and chemical synthesis to restore 24 amino acids, producing Humatrope and Protropin (1985).