Biotechnology

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BIOTECHNOLOGY IN HUMAN WELFARE : 

BIOTECHNOLOGY IN HUMAN WELFARE Dr.Chandrashekhar G Patil M.Sc.,Ph.D. Associate Professor and Course coordinator in Genetics Department of Botany Karnatak Science College, DHARWAD-580 001. Patil_cg@yahoo.com Patil.cg1970@gmail.com Presented at K.Sc.C.D. during training programme for school science teachers of Kundagol Taluk on 09-01-2010.

Introduction : 

Introduction The term was coined by KARL ERKEY- a Hungarian engineer in 1919. It is a fusion of two words BIOLOGY + TECHNOLOGY. The term is very recent (20th century) but not the technology (prehistoric-5000 BC). The technology is as old as Human. Our grand-grand mother is a good “Biotechnologist”. Technology boosted in 1970’s with the discovery of DNA, gene and REN’s.

Definitions : 

Definitions “The technology involved in utilization of → biological entities (micro organisms, cells of higher organisms-either living or dead), → their components or constituents (enzymes), In generating some products which are useful to enhances the quality of human life”.

Scope : 

Scope Biotechnology has emerged as an area of studies and research. It has potential impact on virtually all domains of human welfare, ranging from food processing, protecting the environment and to human health. As a result, it plays a very important role in employment, production and productivity, trade, economics and economy, human health and quality of human life throughout the world. Biotechnology has made marked contribution to human welfare in areas like human health, animal health, agriculture, forestry, horticulture and floriculture, animal husbandry and diary, environment, fisheries and aquaculture…….. In addition to this, marked contributions are also made in renewable energy and fuels, chemicals and biochemicals, population control, food processing and beverages, mining, crime detection and disputed parentage……

Biotechnology and its related areas : 

Biotechnology and its related areas

Economics : 

Economics Biotechnology seems to have an unlimited range of valuable and useful products and services concerned with every aspect of human existence. There are about 325 biotechnology companies in India only, which are mainly involved in production of vaccines, monoclonal antibodies, human hormones like Insulin, HGH, FSH, Interferon etc. In India, about 76% of the Biotechnology revenue is contributed by Bangalore based BIOCON and Pune based Serum Institute and Bio-pharma.

Present thrust areas of Biotechnology are Gene therapy, Stem cell culture, Cloning, Edible vaccines and other medicines.

Some of the India Biotech companies : 

Some of the India Biotech companies 1. Biocon 2. Nicholas Piramal India Limited 3. Dr Reddy's Laboratories 4. Serum Institute of India 5. Wockhadrt Limited 6. Glaxo SmithKline 7. Indian Immunologicals 8. Bharat Serum 9. Krebs Biochemicals and Industries Limited 10.Wipro Health Sciences 11.Shantha Biotechnics Ltd. …..list continues

Biotechnology Products : 

Biotechnology Products Preparation of Wine, Vinegar, Curd, Leavened bread, Yoghurt, Cheese etc., through fermentation by using microorganisms. Butanol and Acetone from starch by bacterium-Clostridium acetobutylicum. Pencillin-a powerful antibiotic from Pencillium notatum (a fungus). Bt-cotton (Bacillus thurengenesis). Flavour-Sever tomato (antiscence RNA technology). Transgenic plants and animals (gene cloning). Human insulin (E.coli). ……continued

? : 

? How these goals of biotechnology achieved ? How the products of biotechnology are produced?

r-DNA technology in Biotechnology : 

r-DNA technology in Biotechnology Central Dogma of Molecular Biology r-DNA Technology Requirements for r-DNA technology Application in Biotechnology Techniques in r-DNA technology Transgenic Plants Transgenic Animals

Central Dogma of Molecular Biology : 

Central Dogma of Molecular Biology DNA RNA Polypeptide (Protein) Replication Tansecription Reverse Transcription (Only in retroviruses) Translation Nucleus Cytoplasm

Slide 16: 

Genetic Information Processing in Eukaryotes

Definition of recombinant DNA : 

Definition of recombinant DNA Production of a unique DNA molecule by joining together, two or more DNA fragments not normally associated with each other. - DNA fragments are usually derived from different biological sources

Development of molecular biology : 

Development of molecular biology Early research on prokaryotic genetics and the development of molecular techniques has led to a new discipline called MOLECULAR BIOLOGY “Tools” have been developed (and still continue to be modified/improved) to enable scientists to examine very specific regions of the genome or genes.

Era of Recombinant Proteins (Since 1980s) : 

Era of Recombinant Proteins (Since 1980s) Target DNA Host cells Product + Plasmid (E. coli, other bacteria or Protein Animal cells) (drugs,industrial enzymes, animal hormones) Gene cloning Transformation Harvesting

Requirement for Recombinant DNA Technology : 

Requirement for Recombinant DNA Technology A. Restriction Endonucleases B. Target DNA C. Vector D. Host E. An Ideal Technology to Harvest GM Products

Slide 21: 

A. Restriction Endonucleases

Origin and Function : 

Origin and Function Bacterial origin = enzymes that cleave foreign DNA Named after the organism from which they were derived EcoRI from Escherichia coli BamHI from Bacillus amyloliquefaciens Protect bacteria from bacteriophage infection Restricts viral replication

Slide 23: 

HOWEVER Bacterium protects it’s own DNA by methylating those specific sequence motifs

Availability : 

Availability Over 200 enzymes identified, many available commercially from biotechnology companies

Types of Restriction Enzymes : 

Types of Restriction Enzymes Type I Cuts the DNA on both strands but at a non-specific location at varying distances from the particular sequence that is recognized by the restriction enzyme Therefore random/imprecise cuts Not very useful for rDNA applications

Types of Restriction Enzymes : 

Types of Restriction Enzymes Type II Cuts both strands of DNA within the specific sequence recognized by the restriction enzyme Used widely for molecular biology procedures DNA sequence analysis Most preferred in r-DNA technology

Properties of RENs : 

Properties of RENs Reads the same in both 5’3’ and 3’5’ direction on both strands = Palindromic Sequence -MALAYALAM or GAATTC CTTAAG * Some enzymes generate “blunt ends” (cut in middle) - GAATTC product GAA TTC CTTAAG CTT AAG Others generate “sticky ends” (staggered cuts)

Action of RENs : 

Action of RENs

Restriction Digestion : 

Restriction Digestion

Slide 30: 

B. Target DNA

Target DNA synthesis : 

Target DNA synthesis Desired DNA with specific product to be cloned Isolated from any source or artificially produced by using REVERSE TRANSCRIPTASE Molded in to manageable size with RENs

Target DNA synthesis… : 

Target DNA synthesis…

Target DNA synthesis…. : 

Target DNA synthesis…. Isolate m-RNA, produce c-DNA by using unique property of RESTRICTION ENDONUCLEASES

Slide 34: 

C. Vector

Vector : 

Vector Vehicle used to transport TARGET DNA from its source to destination (Host) Vector may be Prokaryotic or Eukaryotic in origin It is a small double stranded DNA usually circular

Properties : 

Properties Should have Antibiotic resistant genes and/or other selectable markers enable identification of cells that have acquired the vector construct. Some vectors contain inducible or tissue-specific promoters permitting controlled expression of introduced genes in transfected cells or transgenic animals.

Commonly Used Vectors : 

Commonly Used Vectors Bacterial Plasmids (pBR 322, 327., pUC 18,19 etc.) Phage DNA (l) Accommodates ~15kbp of foreign DNA Eukaryotic DNA Segments (ISE, Transposons) Episomes (Eukaryotes) Cosmid (COS site from l + Ori C from plasmid) intake 35-45 kbp YAC (Intake 1Mb (1kbp = 103 bp) with ARS BAC HAC (Developed in 1997 – synthetic, self-replicating) ~1/10 size of normal chromosome. etc.

Choice of vector : 

Choice of vector Depends on nature of protocol or experiment Type of host cell to accommodate rDNA Prokaryotic Eukaryotic

Plasmid vector : 

Plasmid vector Covalently closed, circular, double stranded DNA molecules that occur naturally and replicate extra chromosomally in bacteria Many confer drug resistance to bacterial strains Origin of replication present (ORI)

Agarobacterium tumefaciens : 

Agarobacterium tumefaciens

Plasmid vector…. : 

Plasmid vector…. Examples pBR322 (Plasmid constructed by Boliver and Roudrigze) One of the original plasmids used Two selectable markers (Amp+ and Tet+ resistance) Several unique restriction sites scattered throughout plasmid (some lie within antibiotic resistance genes = means of screening for inserts) ColE1 ORI

pBR322 : 

pBR322 Description * pBR322 Plasmid DNA is used to create new cloning vectors and for cloning DNA fragments. It can also be digested for molecular weight markers. pBR322 contains the ampicillin and tetracycline resistance genes.

pBR322 chromosome map : 

pBR322 chromosome map

pUC18 : 

pUC18 pUC18 (Plasmid of University of California) Derivative of pBR322 Advantages over pBR322: Smaller – so can accommodate larger DNA fragments during cloning (5-10kbp) Higher copy # per cell (500 per cell = 5-10x more than pBR322) Multiple cloning sites clustered in same location = “polylinker”

pUC18…. : 

pUC18…. Interruptable gene encoding for enzyme beta galactosidase (lacZ) Polylinker resides in the middle Enzyme activity can be used as marker for gene insertion Disrupted gene = nonfunctional Intact gene = functional Media containing XGAL chromagenic substrate used (blue colonies = intact; white colonies = disrupted) Amp resistance gene still present (= beta lactamase), Tet resistance gene omitted

pUC 18/19 Chromosome map : 

pUC 18/19 Chromosome map

Slide 47: 

D. Host

Host cells for recombinant protein production : 

Host cells for recombinant protein production • Bacteria (Used for Human Insulin Production) – E. coli • Fungi (Used for Production of Mycotoxins) – Yeast * Molds • Insect Cells (GFP) • Mammalian Cells (Increased Production of Milk & Meat) • Transgenic Animal (For increased meat/wool Production) • Transgenic Plant (Bt cotton, Vit-D, Carotinoids etc.)

Slide 49: 

E. Technology to Harvest GM Products

Slide 50: 

Development of suitable BIOREACTERS to Harvest GENETICALLY MODIFIED Products

Common Steps Involved in rDNA Technology : 

Common Steps Involved in rDNA Technology 1. DNA molecules (both Vector and Target) are digested with RENs which reduces the size of the fragments  Renders them more manageable for cloning purposes

Common Steps Involved in rDNA Technology…. : 

Common Steps Involved in rDNA Technology…. 2. These products of digestion are to be mixed in presence of an adhesive enzyme (DNA Ligase).  Enables integration of desired DNA fragment to the Vector DNA.

Common Steps Involved in rDNA Technology…. : 

Common Steps Involved in rDNA Technology…. 3. Transformation of rDNA in to the host by making wall of the host permeable by using Calcium Chloride → Enables rDNA enters in to Host cell

Slide 54: 

Each cell receiving rDNA = CLONE May have thousands of copies of rDNA molecules/cell after DNA replication - As host cell divides, rDNA partitioned into daughter cells

Transformation : 

Transformation

Slide 56: 

4. Population of cells of a given clone is expanded, and therefore so is the rDNA. Amplification DNA can be extracted, purified and used for molecular analyses Investigate organization of genes Structure/function Activation Processing Gene product encoded by that rDNA can be characterized or modified through mutational experiments

Product Harvesting : 

Product Harvesting Vectors able to survive under antibiotic selection are amplified in bacterial hosts by autonomous replication Plasmid DNA containing the gene of interest is purified from large scale cultures

Applications : 

Applications Gene isolation/purification/synthesis Sequencing/Genomics/Proteomics Polymerase chain reaction (PCR) Mutagenesis Expression analyses (transcriptional and translational levels) Restriction fragment length polymorphisms (RFLPs) Biochemistry/ Molecular modeling High throughput screening (Micro array technique) Gene therapy

Applications…. : 

Applications…. Recombinant Vaccines Genetically modified crops Biosensors Monoclonal antibodies Cell/tissue culture Xenotransplantation (Transplantation of organs like kidney, heart, eyes etc.) Bioremediation (Waste cleaning through biological organisms) Production of next generation antibiotics Forensics Bioterrorism detection

Gene Transformation in Plants : 

Gene Transformation in Plants

Plant Tissue Culture : 

Plant Tissue Culture Rooting Callus Shoot induction

Plant Tissue Culture…. : 

Plant Tissue Culture….

Transgenic Plants : 

Transgenic Plants

Transgenic Animals : 

Transgenic Animals

Transgenic Animals…. : 

Transgenic Animals….

Human Genome Project (HGP) : 

Human Genome Project (HGP) Entire human genome has been sequenced (April 2000) Project began in 1990 – Joint Venture Human Genome Organization (HuGO) (USA, UK, France, Japan mainly) Still the total genome is not sequenced because of Genome complexity

Some Basic Molecular Techniques : 

Some Basic Molecular Techniques Polymerae Chain Reaction (Molecular Photocopier) -In-vitro multiplication of desired gene or any DNA fragment.

Some Basic Molecular Techniques…. : 

Some Basic Molecular Techniques…. Agarose Gel Electrophoresis Separation of DNA fragments based on size, charge and shape differences Standardized MW markers run on the same gel for size comparison Single and double digests can together aid in the construction of a genetic map

Slide 70: 


Gene Cloning : 

Gene Cloning

Southern blotting : 

Southern blotting Technique developed by Ed Southern used for variety of purposes Procedure: 1. DNA is digested with restriction enzymes and separated by agarose gel electrophoresis (may be photographed if needed) 2. Gel is treated with NaOH to denature DNA  ss DNA

Southern blotting…. : 

Southern blotting…. 3. DNA is transferred from gel to a DNA-binding filter (e.g. nitrocellulose or nylon membrane) using capillary action Gel sits on a sponge wick. Paper towels absorb rising buffer. Buffer passes through the membrane but not the DNA. DNA binds to membrane

Southern blotting…. : 

Southern blotting…. 4. DNA is “fixed” by baking membrane at 80oC or UV cross-linking 5. The membrane is incubated with ss-nucleic acid probe  binds to DNA is complementary. Remainder washed off. 6. Autoradiography or chemiluminescence (dep. on probe)

Slide 76: 

Legend: Legend:

Slide 77: 

Thank you

Slide 78: 

QUESTIONS?