BIOTECHNOLOGY(Karnataka PUC - Biology) DR. M. JAYAKARA BHANDARY Associate Professor of Botany GOVERNMENT COLLEGE, KARWAR
Definition• BT is any technique that uses living organisms or their parts to make or modify products, to improve plants or animals, or to develop micro-organisms for specific uses.• BT is the application of scientific and engineering principles to the processing of materials by biological agents to provide goods and services
BRANCHES & SCOPE OF BT• Recombinant DNA technology or Genetic Engineering: Genetically modifying organisms to create new variety of microbes, plants and animals. Ex. Insulin producing bacteria, Golden Rice, Transgenic cattle, etc.• Tissue Culture: Many plants from small piece of tissue• DNA fingerprinting: Identifying criminals, real parents• Gene Therapy: Disease treatment by gene alteration
GENETIC ENGINEERING• Modifying organisms genetically, by transferring genes from other organisms using Recombinant DNA technology• Creating organisms with new genes – Genetically modified Organisms (GMO’s) or transgenic Organisms.• Addition or Modification of characters
TOOLS FOR GE 1. vectors• DNA molecules that transport foreign genes into a cell or organism.• Ex. Plasmids - circular, small, independently replicating DNA molecules of certain Bacteria• Artificially Designed –• Ex. pUC 18 (plasmid designed in University of California)• pBR 322 (plasmid designed by Boliver & Rodrigues)
2. RESTRICTION ENZYMES (REN)• DNA cutting enzymes – DNA scissors• Cut DNA into double stranded segments at specific regions called Palindromes ( sequence of 4-8 base pairs reading the same in both directions).• Some produce BLUNT ENDS• Some produce STAGGERED OR STICKY ENDS
3. DNA LIGASES• Enzymes that join or ligate the sugar- phosphate backbones of two DNA fragments by forming phosphodiester bonds.• Ex. T4 ligase• Used to join two DNA pieces cut by REN (Vector and gene)• my videosRestrict_enzymes.mov
4. Host cell• Cells into which the genes are transferred.• Bacterial cells are most suitable because they are easy to grow, rapidly.• Common homultiply st – Escherichia coli.
5. BIOREACTORS• Large vessels specially designed to grow genetically modified microbes or cells in large scale and to harvest the product of commercial value• Up to 5,00,000 litres
RECOMBINANT DNA TECHNOLOGY (rDNA technology)• Recombinant DNA – DNA produced by combining two or more segments of DNA taken from different sources.• Recombinant DNA Technology – technique of constructing a recombinant DNA• Important in GE
STEPS IN rDNA TECHNOLOGY1.OBTAINING THE GENE – isolation from cells/from gene libraries/artificial synthesis 2. VECTOR SELECTION - suitable vector selected 3. RESTRICTION DIGESTION – Both gene and vector cut by the same REN to produce sticky ends (Slicing)4. LIGATION – Gene and vector DNA with sticky ends mixed in the presence of DNA ligase. They join and form Recombinant DNA (splicing) 5. TRANSFER INTO HOST CELL – Recombinant DNA is inserted into suitable host cell 6. MULTIPLICATION & EXTRACTION OF PRODUCT – Genetically modified cell multiplied (Cloning), product harvested
ENJOY AN ANIMATION OF rDNA TECHNOLOGY• my videosPLASMIDCLONING.AVI
APPLICATIONS OF rDNA TECHNOLOGY• Production of GMO’s• Production of r Proteins – insulin, growth hormone, Blood clotting factors, etc• Gene therapy• Recombinant vaccines
DNA FINGERPRINTING• Technique which helps in the establishment of identity of a person or organism based on uniqueness of DNA sequence• Developed by Alec J. Jeffreys – 1985• Based on regions of DNA called Variable Number Tandem Repeats (VNTRs)• Uses: Identification of criminals, solving parental disputes, identifying any biological materials.
Steps in DNA fingerprinting 1 . DNA EXTRACTION FROM TISSUE SAMPLES 2. RESTRICTION DIGESTION – cutting DNA into double stranded fragments 3. ELECTROPHORETIC SEPARATION – double stranded DNA fragments separated according to size by electrophoresis on a gel slab4. SINGLE STRAND SEPARATION – double stranded DNA separated into single stands by treating with an alkali 5. SOUTHERN BLOTTING – DNA bands from gel slab transferred to nitrocellulose sheet 6. PROBE HYBRIDISATION – DNA treated with radioactive DNA probes7. AUTO RADIOGRAPHY – X-ray photography of nitrocellulose sheet to obtain DNA fingerprints. 8. COMPARISON OR MATCHING OF DNA FINGERPRINTS
• my videos3ESDSGEL.AVI• my videosSOUTHERN.EXE
GENE THERAPY• Treating diseases by manipulating the genetic material of the patients.• Addition of healthy gene in the place of defective gene by recombinant DNA technology.• Genetic diseases may be cured• Viruses are used as vectors
TYPES OF GENE THERAPY Ex vivo gene therapy • Gene is added to cells separated from body (outside the body). • Ex. ADA deficiency/ SCID In vivo gene therapy • Gene transferred directly to cells inside the body. • Ex. Cystic fibrosis
Somatic gene therapy• Only somatic or body cells are used for gene therapy.• Effect short lived, not inheritedGermline gene therapy• Germ cells (eggs, sperms, zygotes) used for therapy.• Effect permanent, inherited. Not permitted at present for ethical reasons.
MONOCLONAL ANTIBODIES• Antibodies are protein molecules produced by B-lymphocytes during infection/ antigenic stimulation.• Pure antibodies with predetermined specificity which can react with only a single type of antigen are called monoclonal antibodies (MAB’s)• Polyclonal AB’s react with many different antigens
MAB production• By HYBRIDOMA TECHNOLOGY of Georges Kohler & Cesal Milstein, 1975.• Hybridoma cells – formed by fusion of antibody forming B lymphocytes and tumour causing Myeloma cells.• Two properties – Divide continuously and produce antibodies.• Hybridoma cells cultured – MAB’s produced.• Uses: Blood typing, Diagnostic techniques like ELISA, etc
HUMAN GENOME PROJECT• Genome – All genes in the haploid set of chromosomes of an organism.• Human genome – genes in 24 chromosomes (22 autosomes + X & Y chromosomes)• HGP: Project to study the complete sequence of bases of the human genome• 1990 – 2003, 3 billion dollars
OUTCOME OF HGP• Total genes in human genome – 30,000• Total base pairs – 3164.7 million• 99.9% genes same in all humans• Only 2% gene s actually code for proteins.• Function of 98% genes unknown. Junk DNA• Average size of human genes – 3000 base pairs• Largest human gene – dystrophin gene- 2.4 million base pairs.• Max. genes in Ch. 1 (2968). Smallest in Y ch. (231)
Uses of HGP• Complete understanding of Human genetics• Discovery of disease related genes , Helps in developmemt of gene therapy methods. Ex. Cystic fibrosis, cancers, etc.• Development of gene diagnosis methods• my videosLife.mpg
IMPROVEMENT OF CROP PLANTS• BREEDING TECHNIQUES: Production of new improved crop varieties. – Hybridisaton – crossing genetically different plants • Intraspecific, interspecific, intergeneric. - Mutation breeding - Selection – Mass selection, Pureline selection, Clonal selection. - Polyploidy breeding
PLANT TISSUE CULTURE• Technique of growing isolated plant tissues or cells in an artificial medium under aseptic conditions.• Plant cells TOTIPOTENT. Each cell can Multiply and develop in to new plants when cultured.• Growth medium: Nutrients, vitamins, hormones, agar
STEPS IN PLANT TISSUE CULTURE INOCULATION TRANSFER TO OPEN CALLUS FORMATION HARDENING/ ORGANOGENESIS ACCLIMATISATION
USES OF PLANT TISSUE CULTURE• Micropropagation• Disease free plant production• Multiplication of superior/elite plants• Somatic hybridisation/cybrid production• Transgenic plant production• Secondary metabolite production
TRANSGENIC PLANTS• Genetically modified plants which contain one or more foreign genes artificially transferred to their cells.• Transgenes – Transfection – transgenesis• Vector mediated transfer – Ti plasmid (Agrobacterium tumefaciens), Ri plasmid (A. rhizogenes)• Direct gene transfer – micro-injection, gene gun, electroporation
GOLDEN RICETransgenic rice produced by IngoPotrychus & Peter Bayer – 1999..3 genes for beta carotene (Vit. Aprecursor) production transferred torice plant.Beta carotene produced in endospermof rice – golden yellow ricePrevents Vit. A deficiency problems
GOLDEN RICE PRODUCTION• Isolation of genes: 2 from daffodil plant ( Psy gene – Phytoene synthase, Lyc gene – lycopene cyclase), 1 from Erwinia uredovora ( crt 1 gene – Phytoene desaturase ).• Insertion of genes to rice embryo cells: Using Ti plasmid• Transgenic plant production: Genetically modified embryos developed into rice plants.
USES OF TRANSGENIC PLANTSProducing plants with better nutritionalquality. Ex. Golden riceProducing plants with disease/pestresistance. Ex. Bt cornProducing plants with new characters. Ex.Vaccine producing plants.
IMPROVEMENT OF ANIMALSMating: Crossing two selected parent animalsto produce superior progenyArtificial Insemination: Semen (sperms) ofsuperior males artificially placed in thereproductive tract of female animals to effectfertilisation. More females can be fertlised bysuperior males. Ex. Cattles, horses.Multiple Ovulation and Embryo transfer(MOET): Superior females induced to producemore eggs per cycle, Fertilised to form manyembryos, Embryos collected and transplantedto surrogate mothers. Useful to multiplysuperior females.
STEM CELL CULTURE• Immature cells with the potential to develop into many different cells of animal body.• PLURIPOTENT/MULTIPOTENT• Adult stem cells & embryonal stem cells.• Can be cultured to form muscle cells, nerve cells, etc for transplantation therapy.• Cultured stem cells can also be used for genetic modification (Bone marrow cells).
TRANSGENIC ANIMALS• Genetically modified Animals with one or more foreign genes.• Gene transfer by viral vectors & Micro-injection method.• Genes introduced to eggs or early embryonal cells, induced to form zygotes, transplanted into surrogate mothers.
TRANSGENIC CATTLE PRODUCTION (Nuclear transfer technology)
USES OF TRANSGENIC ANIMALS• Production of recombinant proteins . Ex. Human growth hormone, Clotting factor.• Models to study human diseases: Oncomouse,• Production of improved animals: Fast growing pig, cattle, etc.
HAZARDS & SAFEGUARDS OF GEDangerous GE research strictlyorganisms – supervised.pathogens – Bio Permission of rDNAterrorism. advisory committeeFood from GMO’s essential.dangerous to health GM organismsGM crops also kill cannot be releaseduseful insects – to open withoutbutterflies proper testing.Modifying human Labelling GM foodsgerm cells – against compulsory.nature, unethical Human GE banned.