U. Satyanarayanaae | 11 Manipulation af Gene Expression in Host Cells 137, Introduction to Biotechnology 12 Human Genome Project 148 Piece ani 5 TI SGIUNWTERENEET: | Medicat/Pharmaceutical —— Biotechnology Introduction to Molecular Biotechnology in Healthcare) Biology poled 13 Gene Therapy 157 PAA PUN coraitanetg SaeaeaE | 14! Seth ese Clagbisheed test DNA-Replication, Recombination, Forensics and Repair 23 15 Pharmaceutical Products of DNA Transcription and Tanslation 38 Technology 189 Fpl Sioni oe GEE Epracion aise 16 Recombinant Vaccines 199 as fen 17 Monoclonal Antibodies [section (Hybridoma Technology) 213, ” AB Assisted Reproductive Technology 227. Genetic Engineering/ Recombinant DNA Technology | LAA Introduction to Genetic Engineering 75 | Microbial/Tndustrial Basic Techniques in Genetic Engineering 92 Biotechnology Polymerase Chain Reaction 19 Bioprocess/Fermentation Technology 239. (DNA Amplification) 112 | 20 Dovnsteam Procesing 270 jene Libraries 120 les Enzyme Technology 2817 lirected Mutagenesis and Protein 22 Biotransformation 306 129 123 Microbial Production of Organic Solvents 3112 moti Peco fOnaric Ads 318 49 Cert Engen a mee Ce 26 Microbial Production of Amino ‘Acids 344, 50. Applications of Plant Teansformatig 2g ach kc ee ee ae gat tees 1 Bee romaine Bac in Pa 29 Single-Cell Protein, and Mushrooms 373 $3. Molecular Marker-Aided Plant Bresdl 29 sie al Pr Foca ani | TSS TS rae . 31. Biomass and Bioenergy 393 Environmental Biotechnole 32 Mirai! Mining and etl Biotechnology 400 | 54. Environmental Pollution — General 155 Air Pollution and Control 669) [SE 56 Water Pollution and Sewage 157 Sewage/Waste Water Treatment ‘Snimal Cell/Animal 58. Sludge and Sold Wastes-reatmel oes Dispos 708 ee logy ‘59 Biodegradation and Bioremedi 23. Arima Cel Calture— Fundamental Facies | 60 iota Evronmeta Problems td Applications 407 24 Cate infor Anal Cells 415 Beis Coe OH Se | Biotechnology and Sock 36 oe utre and Cell Uns 437. | 61. Bitchnolony— Society, Rk FF Rare att Cukure General Considerations | Pateting 739 and Scilewp 450 38 Call Viability and Cytotoxicity 459 Ea cstonuoenendiod Changs 63 locas ao" Tceme tenn oe Biotes Engineeing 468 62. Call—The Basic Unit of ie 41 Wamsgeic Animals 480 @ Micwornisns 734 64 Bioorganic and Biophysical iE echemisty Tools 758 —___ 65 Biomolecules 71 Piant/Agricultaral Biotechnology 66 Exzymology 786 i seine Paar. — | 2 Metobene 8 43 Plant Tesue Culture Media 517 fae ye 44 Prxoplst Cure and Somatic Be aes yitizaion 523 . 45 Production of Haplod Plans 538 {46 Somacloral Varaions 546 0 eacdin oitmedsaers 9902 (OY |S 48 Germplasm Conservation and Abbreviations 852 Cryopreservation 565 Index 855 AppendicesOrganization of DNA inthe cll Structure of RNA Introduction to Biotechnology Types of RNA, 1 The Scope of Biotechnology Sede nd toy eecrnoey 3 DNA-Replcation, Recombination inition) of biotechnology da Pe History of biotechnology Replicati oe DNA 2B Biotechnology —a mulidlscipinary ate s ee ‘plication in prokaryotes 2 Replication in eutaryotes a Commercalizaton of biotechnology = = E ee Public perception of biotechnology oe The fate of biotecnlogy Cal CEE OSE 29 Organization ofthe book Telomeres and telomerase 30 Telomere in senescence and cancer a1 SIRE Recombination 31 Homologous recombination 31 'Non-fomologous recombination 3 2. DNA and RNA — Composi Damage and repair of DNA 33 and Structure 11 Types of DNA damages u Nucleotides 11 Mutations Mu Structure of nucleotides 11 Repair of ONA 5 Structure of DNA t4 Deets in DNA repair and cancer 37 DNA double helix 4 (Other types of DNA structure 7 Introduction to Molecular Biology 4 Transcription and Translation 38 The size of DNA molecule-units Genome 38 «feng 17 Fansrptome 38 Denatration of ONA stands Proteome 2 oCONTENTS IN DETAIL ‘Transcription Transcription in prokaryotes Transcription in eukaryotes Postsranscrptional modifications Cellular RNA contents Reverse transcription Translation Genetic cade Protein biosynthesis Requirement of the components ‘Activation of amino acids Protein synthesis proper Initiation of translation Elongation of translation Termination of translation Inhibitors of protein synthesis CChaperones and protein folding Postransational modifications of proteins Protein targeting ‘Mitochondrial DNA, transcription and translation 5 Regulation of Gene Expression Gene regulation — general ‘The operon concept Lactose (lac) operon “Tryptophan operon Gene expression in eukaryotes Chromatin structure and gene ‘expression Enhancers and tissue-specific ene expression ‘Combination of DNA elements and proteins in gene exoression otis in proteins and gene expression Gene regulation in eukaryotes Methods to study gene expression/regulation Gene analysis by T-DNA and transposon tagging Methods to study protein-protein interactions Phage display Yeast two-hybrid system Genetic Engineerin, DNA Technology 6 Introduction to Genetic Brief history of recombinant DNA. technology Molecular tools of genetic e Restriction endonucleases-ONA DNA ligases-DNA joining en Alkaline phosphatase DNA modiving enzymes Host cells — the factories of el Prokaryotic hosts Eukaryotic hosts Vectors —the cloning vehicles Plasmids Bacteriophages Coemide Atficial chromosome vectors Shuttle vectors Choice of a vector Methods of gene transfer “Tansformation Conjugation Electroporation Liposome-mediated gene ta “Transduction Direct transfer of DNA. Gene cloning strategies Cloning for genomic DNA Genetic engineering of plants Genetic engineering guide Asilomar recommendations NIH guidelines The future of genetic engi 7 Basic Techniques in Genetic Engineering Agarose gel electropho Pulsed-field gel electCovrenrs mi Dera il SS Contour-clamped homogeneous electrical- field electrophoresis (CHEF) Polyacrylamide gel elecuophoresis (PAGE Other uses of gel electrophoresis Isolation and purification of nucleic acids Patification of cellular ONA Purification of plasmid UNA Patcation of mRNA Isolation of chromosomes Fluorescence-activated cell sorting Collection of chromosomes with identical sizes Nucleic acid blotting techniques Southern bloting Northern lating Dotlating Western blotting Autoradiography Colony and plaque blotting DNA sequencing ‘Maran and Gilbert technique Dideoxynucleotde method Bacteriophage M, as a cloning and DNA sequencing vector DNA sequencing by primer walking Chromosome walking in DNA sequencing ‘Automated DNA sequencing Alternative methods of DNA sequencing Pyrosequencing DNA chips (microarrays) Chemical synthesis of DNA, The phosphoramidite method Synthesis of genes 8 Polymerase Chain Reaction (DNA Amplification) Techrique of PCR Sources of DNA polymerase Key factors for optimal PCR Variations of PCR Nested PCR Inverse PCR Anchored PCR 93 3 94 94 94 95 9% 96 96 Oa 97 99 99 100 100 100 tor Reverse transcription PCR ‘Asymmetric PCR Realtime quantitative PCR Random amplified polymorshic DNA (RAPD) “Amplified fragment lenth polymorphism (AFLP) Rapid amplification of CDNA ends (RACE) Applications of PCR PCR in clinical diagnosis PCR in DNA sequencing PCR in gene manipulation and expression studies PCR in comparative studies of genomes PCR in forensic medicine PCR in comparison with gene cloning 9 Gene Libraries Creating 2 gene library PCR as an alternative ta genomic library constuction Complementary DNA libraries Screening strategies Screening by DNA hybridization DNA probes Screening by colony hybridization Screening by PCR Screening. by immunological assay Screening by protein function 10 Site-directed Mutagenesis and Protein Engineering Strategies to improve in vitro activities of enzymes ligonucleotide—directed mutagenesis Casettee mutagenesis, PCR-amplified oligonucleotide-directed tagenesis Protein engineering Increasing the tailty and biological activity of proteins Addition of dsulidle bonds Changing asparagine t other amino acids Reducing the ire sulyay groups Single amino acid changes us m5 116 116 17 18 118 118 n9 119 ug 119 19 120 120 ma 13 125 125 126 126 128 128 128 129 19 10 BI 132 133 133 ta 133 133 134(i) CONTENTS IN DETAIL ectiheet pore teams 133. Raheem Fee mienmaks pre ome ‘con ge Ca ‘ones 8m A ieee iain ran aca ch eae iit tone ee eee es seca nlc an code a ihe ancy Cine oo ae Host cel, 197 thespy fara iia ne er cog Mam ar ies orsteen eerste ‘hey sot aes 130 Thao eel eee Bite poe boat Raves oy lane ie a. here expressing cloned genes 140 Gene delivery by viruses: Other yeast expression systems Gene delivery by non-viral Insect cell expression systems Gene therapy strategies for ¢: onan eee Gere heap or ADS er eae pee haere ll auiatiMaevatiaeen hee kant ee ae free happy a cle oleae pete Aare sero ane 12 Human Genome Project Chimeric oligonucleotides in gene: rite st of tren te ae rc janet icone reer cla ore fihoraot ss Genre al ‘Approaches for genome sequencing The future of gene therapy Be ooce senaon summarised 14 DNA in Disease Diagnosis Fo ct ohana einen aha wore eae ee Nucleic acid hybridization Ethics and human genome DNA probes ‘The DNA chip-microaray of F gene probes DNA inthe diagnosis of infectious Medical/Pharmaceutical diseases Biotechnology Tuberculosis Giotechnology in Healthcare) pia Chagas disease 13 Gene Therapy Acquired inmunodeficlencysyndi Approaches for gene therapy (aids)Human papilloma virus lyme disease Periodontal disease DNA probes for ther diseases DNA in the diagnosis of genetic diseases Cystic fibrosis Sichle-cell anemia Duchenne’s muscular dystrophy Huntingtons disease Fragile X syndrome Other tiple repeat diseases Alaheimer’s disease Amyotrophic lateral sclerosis Cancers Diabetes Obesity DNA analysis for ather human diseases Gene banks —a novel concept DNA analysis for environmental ‘monitoring Water quality testing DNA fingerprinting or DNA profiling DNA markers in disease diagnosis and fingerprinting Restriction fragment length polymorphisms (RFLPs) Variable number tandem repeats (WNTRS) Microsatelites (simple tandem repeats) Single nucleotide polymorphisms (NPS) Current technology of DNA fingerprinting 15 Pharmaceutical Products of DNA Technology ‘Human protein replacements Insulin Human growth hormone Clotting factor Vi Therapeutic agents for human diseases Tissue plasminogen activator Interferons 178 178 178 178 179 179 179 179 180 180 180 181 181 tat 1a 183 183 184 108 184 184 185 185 185 188, 188 188 189 189 189 192 194 194 198 195 Conran my Dera Eythropotetin Deoxybonuclease | (DNase Alginate Ivase , Second generation therapeutic proteins (ruteins) Vaccines 16 Recombinant Vac Traditional vaccines Recombinant vaccines — general Subunit vaccines Hepatitis 8 Foot and mouth disease Herpes simplex virus Tuberculosis Meningitis AIDS (Acquired immunodeficiency syndrome) DNA vaccines (genetic immunization) IDNA vaccine and immunity RNA vaccines Plants as edible subunit vaccines Attenuated recombinant vaccines Cholera Salmonella species Leishmania species Vector recombinant vaccines Vaccines against viruses — Delivery of antigens by bacteria 17 Monoclonal Antibodies Principles for creation of hybridoma cells Production of monoclonal antibodies Large scale production of MAbs Human monoclonal antibodies Gonatc engineering strategies forthe production of human-mouse MAbS Production of MAbs in E.coli ‘Second generation monoclonal antibodies ‘Advantages of monoclonal antibodies Limitations of monoclonal antibodies ” 198 198, 198 198 198 199 199 200 201 201 202 203 203 203 204 205 205, 207 207 208 208 210 210 a m a2 m3 23 a4 216 216 27 218 218 219 219(wi) CONTENTS IN DETAIL tions of monoclonal antbodi 219 Diagnostic applications 219 ‘MAbs in biochemical analysis 219 MAbs in diagnostic imaging 220 ‘Therapeutic applications 22 ‘MAbs as direct therapeutic ages 22 ‘MAbs as tating agents in therapy 223 Protein puiieation 2235 Miscellaneous applications 226 Cataltic MAbs atzymes) 226 Antibody fiogerprntng 26 18 Assisted Reproductive Technology 227 Manipuation of reproduction in animals 227 Aificial insemination 207 Embryo wansier 228 Invitro fertilization 230 Embryo cloning 231 ‘Manipulations of reproduction in humans 231 Causes of infertility and application of ART at Invauteine insemination (U0 2 {In vito ierilization and embryo transfer {VF and ET) 22 ‘Methodology of 1VF 232 Success rates of VF 23 The words picture of test tube babies 233 Gamete intrafllopian tansier (GIFT) 233, ‘Zygote inteafallopian transfer ZIFT) 234 Intavaginal culture OVC) 234 ‘Cytoplasmic transier (CT) 234 Micromanipulation 234 ‘Cryopreservation 235 Assisted hatching (AH) 235 Preimplantation of genetic diagnosis PED) 236 The negative aspects of ART 236 Sarma Microbial/Industrial Biotechnology 19 Bioprocess/Fermentation Technology 239 Bioreactos/Fermenters 239 “Types of bioreactors 239 ‘A conventional bioreactor — common features ‘Operation of a conventional bioreactor Sterilization Inoculation and sampling Aeration Control systems Cleaning Solid substrate (solid state) fermentation ‘Media (substrates) for industrial fermentation Substrates used as carbon sources Substrates used as nitrogen sources Sources of growth factors Sterilization of culture media and gases Sterilization of culture media Sterilization of air Isolation of microorgani Preservation of microorganisms Microbial metabolic products —low molecular weight compounds Primary metabolites Secondary metabolites Bioconversions bial metabolic products — high ‘molecular weight compounds Genetic improvement (development) prt ‘Methods of sain development Maration Genetic recombination Principles of microbial growth and culture sytem Batch culture or batch fermentation Fed batch culture or fed-batch fermentation Semi-continuous culture or semi ‘continuous fermentation Continuous culture or continuous fermentation Growth kinetics of microorganisms Classification of fermentation processes The fermentation process ‘Measurement and control of Bioprocess parameters Seale-up Food technology Food preservation 248 245 245 245 245 246 246 247 248 9 m9 250 250 250 251 252 254 28 234 255 257 257 257 258 258 259 259 259 261 261 262 263 264 265 267 268 268 26920 Downstream Processing 270 Stages in downstream processing 270 Salidiquid separation 20 Fettion m Flocculation mm Fitvation m Centiugaion m Release ofinacellular products m3 Cell dsupion on Canceatation 276 ‘vaporation 276 tiguiebiquid exraction 276 ‘Membrane fieation a7 Precipitation a Adsorption ms Puetication by chromatography 278 Formulation 279 Integration of dierent processes 280 21 Enzyme Technology 281 Applications of enzymes 281 Commercial production of enzymes 281 The technology of enzyme producion—genetal considerations 283 Regulation of microbial enzyme production — general considerations 286 Genetic engineering for microbial enzyme production 286 Immobilization of enzymes and cells 288 Methods of immobilization 288 Choice of immobilization technique 290 Stabilization of soluble enzymes 290 Immobilization of cells 233 Etec of immobilization on enayme proven 293 Inmabilzed enzyme reactors 204 Applications oF immobilized enzymes and cells 295 ‘Manufacture of commercial products 295 Inmobilized enzymes and cel — analytical applications 296 Biosensors 27 Types of biosensors 208 ‘lectrachemical biosensors 299 Theaometic biosensors 300 Conreine m Beran (vi) Opiical biosensors 301 Prezoeectc biosensors 302 Whole cell biosensors 302 Inmunobiosensors 303 Aplications of biosensors 304 Immobilized enzymes and cells— therapeutic applications 304 22 Biotransformation 306 “Types of biotransformation reactions 306 Sources of biocaalyts and techniques for biotransfrmation 306 Product recovery in biotransformation. 307 Examples of biotransforation 308 Biotransiormation to produce commercial products 308 Biotransformation of steroids 308 Biotransformaton of antibiotics 308 Bioiransformation of arachidonic acid ‘0 prostaglandins 310 Biaransformation forthe production of ascorbic acid 310 Biotransormation of glycerol to dlinydroxyacetone 310 Bictransformation forthe production of indigo 310 23 Microbial Production of ‘Organic Solvents an Alcohol at Production of ethanol by fermentation 312 ‘Acetone and butanol 315 Glycerol 316 24 Microbial Production of Organic Acids 318 Citic acid 318 Factors inthe regulation of citi acid production 320 Production process for citi acid 21 Surface processes 321 Submerged processes 322 Production of citic acid from alkanes 322 Recovery of citi acid a2 Gluconic acid au(wim) CONTENTS IN DETAIL Lactic acid Acetic acid Ascorbic acid Haconie acid 25 Microbial Production of Antibi Antibiotics — general Aplications of antibiotics Production process of penicilin Production of 6-amino penicilanic acid Cephalosporins Production pracess af cephalosporin Production of 7-aminocephalospocanic acid [New f-lactam technology for production of ACA Aminoglycosides Production process of steptomyein Tetracyctnes Prodiction process of chlortetracycline Prodluction of tetracycline-iferent proces Macrolides Production process of erythromycin ‘Aromatic antibiotics Chloramphenicol Griseofulvin Nucleoside antibiotis Genetic manipulations of Streptomyces Good antibiotic manufacturing practices 26 Microbial Production of ‘Amino Acids Amino acid production — general considerations Commercial applications of amino acids ‘Methods for production of amino acids Stain development for amino acid production L-Glutamie acid Lysine LThreonine L-Phenylalanine 305 326 307 328 329 329 330 330 331 32 334 334 3u4 335 336 336 337 38 339 339 340 340 341 341 341 342 342 Ha 344 344 344 345 6 346 350 352 L-tryptophan Aspartic acid 27 Microbial Production Vitamin By. Commercial production of vita Riboflavin Commercial production of ribola B.Carotene Commercial production of B-carotene Gibberelin-plant growth stimulants ‘Microbial production of gibberellins 28 Microbial Production of Foods and Beverages Fermented Foods Cheese Yoghurt Sauekraut Bread Baker's yeast ‘Other vegetarian foods Sweeteners Thaumatin—a sweet protein ‘Monellia—a candidate for sugar substitute Flavour enhancers 36 Alcoholic beverages 367 Aleoholic beverage production — ‘general aspects 368 Beer 369 Wines 370 ‘Other microbially-derived food products 371 Enzymes in food and beverage industries a71 Enzymes in the preparation of fit juices a7 Diagnostics in food biotechnology a7 Food biotechnology — public acceptance 372 29 Single-Cell Protein, and Mushrooms 373 Microorganisms and substrates used for production of SCP 374Production of SCP from wastes Production of SCP from wood Production of SCP from CO, Production of SCP from sewage Genetically engineered artificial protean eed Mushrooms Production of edible mushrooms +30. Polysaccharides, Polyhydro- ayalkanoates and Lipids Polysaccharides General features of microbial polysaccharides Xanthan Dextran Alginate Scleroglucan Gellan Pollulan Curdlan Polyhydroxyalkanoates PHA-chemisty and properties Polyhydeoxybutyrate (PHB) Biosynthesis of other PHA Biopol —a biodegradable plastic Genetic engineering for PHA ‘production Microbial lipids Production of single-cell ols Microbial rubber Microbial adhesive biopolymer Microbial production of 31 Biomass and Bioenergy ‘Sources and utilization of biomass Production of alcohol from biomass Production of biogas from biomass Process of biogas production (biogas plant Hydrogen —a new biofuel Production of biohydrogen Production of SCP from high enessy sources 375 a78 379 379 379 380 380 381 382 382 383 383 385 385 385 386 386 386 386 386 387 389 300 390 391 391 91 392 392 393 393 394 395 395 397 398 398, Covrenrs i DErAL Energysrich crops ‘The future of biomass 32 Microbial Mi Biotechnology Bioleaching Commercial process of boleaching Bioleaching of copper Bioleaching of uranium Bioleaching of other metals ‘Advantages of bioleaching Biosorption Microbial recovery of petroleum Sa a and Metal Animal Cell/Animal Biotechnology 33 Animal Cell Culture — Fundamentals, Facilities and Applications Facilities for animal cell culture ‘Minimal requirements for cell culture Inirasructre Equipment Culture vessels Use of non-adhesive substrates in tissue culture Contamination, aseptic conditions, and sterilization Aseptic conditions Sterilization Advantages and limitations of tissue culture Applieaions of animal coll cultures ‘Medicapharmaceutcal produets of animal cell cultures Genetic engineering of animal cells and their applications Risks in a tissue culture laboratory and safety Biohazards 34 Culture Media for Animal Cells Natural media 399 399 400 400 401 402 402 403 403 403 408 407 407 408 408 408 408 409 409 409 410 au 412 43 43 414 a5 a5(4) __ CONTENTS IN DETAIL Aificial media Physicochemical properties of culture media Balanced salt solutions ‘Complete culture media Serum Selection of medium and serum Supplementation of the medium with tissue extracts Serum-free media ‘Advantages and disadvantages of serumiree media Development of serum-free media Commonly used serum-free media 35 Cultured Cells—Biology and Character Characteristics of cultured cells Cell adhension Celt proliferation Cell diferertiation Metabolism of cultured cells Initiation of cell culture Evolution and development of cell lines Characterization of cultured cells “Morphology of cells Species of origin of cells Identification of tissue of origin “Transformed cells Identification of specific cell lines Measurement of growth parameters of cultured cells Growth cycle of cultured ces Plating efciency of cultured cells Cell synchronization all enaration by physical mene Call separation by chemical blockade Some highlights of cell synchronization Senescence and apoptosis Cellular senescence ‘Measurement of senescence Apoptosis ‘Measurement of apoptosis 36 Primary Culture and Cell Lines Primary cell culture a5 15 417 47 419 20 420 az al 422 422 423 223 43 404 424 425 425 427 428 4128 429 429 430 430 432 432 433 433 a 434 235 435 435 435 235 436 437 47 Techniques for primary culture ‘Mechanical disaggregation Enzymatic disaggregation Primary explant technique a Separation of viable and non-viable cells ‘Medical ethics and safety measures in culture techniques ell Fines Finite cell lines Continuous cell ines Nomenclature of cell ines Selection of cell ines Maintenance of cell cultures Subculture Monolayer cultures Suspension cultures ‘Stem cell cultures Embryonic stem (ES) cells Epithelial cells ‘Maintenance of stem cells in culture Characterization of stem cells Applications of cultured stem cells 37 Animal Cell Culture — General Considerations and Scale-up Call culture-general considerations Cell quantitation Equipment and medium pH and buffer systems Oxygen Growth kinetics ‘Types of culture processes Otter practical considerations Sealeup Scale-up in suspension Factors in scaling up Stier culture Continuous flow culture Aielift fermenter culture Other systems for suspension culture Scale-p in monolayer Roller botle culture Muitsurface culture Maltaray disks, spirals and tubesCove m Devan tah Microcar culture 456 Tisue modeling a7 Perfused monolayer culture 457 Embryonic stem cll engineering a7 Monitoring of ell growth &S cal cultures to produce in scale-up 458 diferentiated cell 7 Monitoring of suspension cultures 458 Human embryonic stem cell esearch 477 Monitoring of monolayer cultures 458 AL Transgenic Animals 480 38 Cell Viability and Cytotoxicity 459 mporance of transgenic animale general 480 Limitations of in vivo cytotoxicity studies 459 Tranogenic mice 481 ‘Assays for cell viability and cytotoxicity 460 Retroviral vector method 481 Cytotoxicity and viability asays 460 Wicrinjecon method 8} peers 461 Embryonic stem cell method 482 Metabolic assays er ape Ee eect 482 Yeast anifcil chromosome in transgenes 485 Ses 462 Transgenic mice and their applications 485 39 Cell Transformation and Behan oa cattchase ‘4g. The Alcheimer’s mouse 485 Eee The oncomouse 486 Transformation of cells 463 The prosate mouse 486 Gone insaity ee reais “a immortalization 463 bean eR) et _Tasonic mice for human diseases 487 See fteg__TWansgeness in large animals 487 call cloning feg_—_Tanspenic catle 488 ar ee ddeg__‘Warsgenic sheep and goats 480 Stimulation of plating efficiency 465 Tansgenic pis a Suspension cloning 1467 —_Tansgenic chickens 489 Isolation of clones fer er a Position effects 490 40 Organ and Histotypic Cultures, “Asma bioreactors 490 anditisrue:Englncering 468 Dolly — the transgenic clone 490 Organotypic models 468. Cloning of pet animals 491 Organ cultures 4469, ‘Transgenic animals produced by Techniques for organ culture 463 _ cloning fetal cells a eee {70 Teansgenic animals in xenotranspantation 492 Tivee dimensional cultures 471 Pisin xenotansplatation? 492 Oganotypic cultures 472. Wansgenic organisms to interrupt ‘Tiles ghering 72 disease cycles 493 Call source and culture 473 Tansgenic sais 493 ature of cells > 473 Transgenic mosquitoes 493 al onertation 474 Tansgene tsetse les 93 Cell support materials 474 Tansgenc boivorns 493 Design and engineering of tsues 475 Tansgenic meies 493(xy CONTENTS IN DETAIL Plant/Agricultural Biotechnology 42 Plant Tissue Culture — General Benefits of plant tissue culture Basic structure and growth of a plant Conventional plant breeding and plant tissue culture “Terms used in tissue culture Brief history of plant tissue culture “ype of culture Basic technique of plant tissue culture Callus culture Cell culture Isolation of single cell Suspension cultures — growth and subculture Types of suspension cultures Synchronization of suspension cultures Measurement of growth of cultures Measurement viability of cultured cells Culture of isolated single cells (single cell clones) Applications of plant tissue cultures Secondary metabolites in plant culture ‘Applications of secondary metabolites Production of secondary metabolites Selection of cell lines for high yield ‘of secondary metabolites Large scale (mass) cultivation of plant cells ‘Mediarm composition and eiect of nutrients “lietorinduced production of secondary metabolites ect of environmental factors Biowanstormation using plant cell cultures Secondary metabolite release and analysis 43 Plant Tissue Culture Media ‘Major types of media 497 497 497 498 498 498 499 499 500 502 502 502 503 504 505 505 505 506 S07 509 Constituents of media Preparation of media Selection of a suitable mediom 44 Protoplast Culture and Somatic Hybridization Importance of protoplasts and their cultures Isolation of protoplasts ‘Mechanical metnod Fnaymatic method Culture of protoplasts Culture mesa Culture methods Regeneration of protoplasts Sub-protoplass Somatic hybridization Fusion of protoplasts Selection of hybrid cells Identification of hybrid (cells) plants Chromosome number in somatic hybrids yrds Applications of somatic hybridization Limitations of somatic hybridization 45 Production of Haploid Plants In vivo and in vitto approaches Androgenesis ‘Anther culture Pollen (microspore) culture Development of androgenic haploids Factors alfectng androgeness Gynogenesis Identitication of haploids ‘Morphological approach Genetic approach Diploidization of haploid plants (production of homozygous plants) Colchicine treament Endomitosis Applications of haploids Crops developed through anther culture Limitations of haploids 46 Somaclonal Variations Basis of somacional variations 507 528 528 529 5a 53 5a 535 536 537 538 538 539 539 539 540 540 542 543 543 543 543 3B 54a 544 545 545 546 546Isolation of somaclonal variants Without in vite selection With jn vitro selection Factors affecting production of somaclonal varants Applications of somaclonal variations Limitations of somactonal variations Gametocional variations 47 Clonal Propagation (Micropropagation) Technique of micropropagation ‘Mukiplication by axlry buds and nical shoots Muliplication by adventitious shoots Factors afecting micropropagation Organogenesis Somatic embryogenesis ‘Applications of micropropagation Disadvantages of micropropagation Production of disease-free plats Methods to eliminate viruses in plants liminaton of pathogens other than viruses Merits and demerits of disease‘fee plant rodiction Embryo culture Mature embryo culture Embryo rescue Nutritional requirements of embryo cure ‘Aplications of embryo cate 48 Germplasm Conservation and Cryopreservation Insit conservation xsi conservation Cryopreservation “Techniques of cryopreservation Cold storage Low-presure and low-oxygen storage Applications of germplasm storage Limitations of germplasm storage 49 Genetic Engineering of Plants-Methodoogy Gene transfor methods sar 57 57 519 549 351 532 552 553 556 556 356 587 559 559 560 560 562 562 562 562 562 564 565 565 5365 367 569 570 sr sr 572 373 Conrenrs i Dera Vector mediated gene transfer Agrobacteriummediated gene transer Crown gall disease and T: plasmid laity root disease of A. rhizogenes — Ry plasmids 1 plasmid derived vector systems Plan transformation technique using ‘Agrobecterium Virus-mediated gene transfer (plant viruses as vectors) Caulimaviruses as vectors Geminivieuses as vectors RNA plant viruses as vectors Limitations of viral vectors in gene transier Direct or vectoless DNA transfer Physical gene transfer methods Electroporation Particle bombardment (biotic) Microinjection Lipesome-mediated transformation Silicon carbide fibre-mediated Chemical gene transfer methods Polyethylene alycol-mediated! transormation DEAE dextran-mediated transl Calcium phosphate co-precipitation mediated transfer DNA imbibition by celstssues ‘Marker genes for plant transformation Selectable marker genes Antibiotic resistance genes ‘Antimetabolite marker genes Herbicide resistance genes Protliction of marker‘ree transgenic plants Clean gene technology Reporter genes Promoters and terminators Transgene stablliy, expression and gene silencing Scafold attachment regions and gene stability Introns and gene expression Gene silencing Chloroplast transformation Chloroplast engineering(iv) CONTENTS IN DETAIL 50 Applications of Plant Transformation/ Transgenic Plants Resslance to biotic sreses Insect (pest) resistance Resistance genes fom microorganisms Resistance genes fom higher plans Proteinase protease) inibitos lectins Restance genes fom animals Insect resistance though copy are sttegy Virus resistance Fungal and bacterial diseases Pathogenesi-eated (PR) potens Ribosome-inactivain proteins (1S) Phyoalxins Nematode resistance Resistance to abiotic stresses Herbicide restanee Glyphosate resistance Phosphinthvicinresisance Sulfonylareas and imidszoinanes Evironment impact of hesicide- resistant crops Tolerance to water deficit stresses Resistance against ice nucleating bacteria Improvement of crop yield and quality Green revalution Genetic engineering for extended shelie of tus Biochemical changes during tomato Fiponing Genetic manipulation of fut ripening longer shel of ite ae vqetlos Genetic engineering for prevention of eiscooration Genetic engineering for flower pigmentation Genetic engineering fr mal stety Transgenic plans with improved ution Amino acid of seed storage proteins Genetic engineering for imposing palatability of foes 596 596 597 597 600 cot oot 601 601 602 604 605 605 606 605 607 607 607 609 610 610 610 ent 612 612 612 ona on 616 616 67 67 67 618 Golden rice—the provitamin A enriched rice 619 Genetic engineering to increase vitamins and minerals 20 Commercial transgenic crop plants 20 ‘Transgenic plants as bioreactors a 51 Transgenic Plants as Bioreactors 622 Metabolic engineering of carbohydrates 622 Starch on yclodextins 623 Fructans 623 Trehalose a4 Metabolic engineering of lipids 624 Production of shorter chain fatty acids 624 Production of longer chain fay acids» 625 Production of fatty acids with modified degree of saturation 025 Production of rar fatty acids 026 Biodegradable plastics (bioplastics) 626 Genetically engineered plants as protein Factories 028 Approaches for protein production in plans 628 ‘Oleosin patton technology ou Production of indus enzymes in plants. 631 Production of lysosomal enzymes in plants 633 Production of antibodies (plants) in plants 634 Production of vaccines in plans 636 Production therapeutic proteins in plants 637 52 Growth-Promoting Bacteria Plants 639) Biological nitrogen fixation 639 Nitrogen cycle 9 Nivogen fixing bacteria 9 Mechanism of ritrogen fixation 6a Genetic manipulations for nitrogen fixation 641 Genetic engineering of nitrogenase gone 642 Genetic engineering of hycrogenase gene 644 Genetic engineering of nodulation genes 644 Biocontrol of phytopathogens oa Siderophores Antibiotics EnzymesBiofertlizers oss Symbiotic nitrogen fers 646 Asymbiotc nitogen fiers 646 Phosphate solubilizing bacteria 646 Organic ferilizers 647 Benefits of biofetilizes or Limitations of bieriizens 53 Molecular Marker-Aided Plant Breeding oa Molecular markers 630 Markers based on DNA hybiczation 649 Mathers based on PCR amplification 650 Molecular marker assisted sle 632 Molecular breeding 632 Quantitative trait lo 632 ‘Arid and semi-arid plant biotechnology 653, Greenhouse and greenhome technology 653, Environmental Biotectmology 54 Environmental Pollution — General. 637 Enviroament-basic concepts 657 ‘Atmosphere 657 Hydrosphere 657 Lithosphere 658 Biosphere 658 Environmental pollution-sources and nature 658, Sources of pollution 658 Nature of pollutants 658 Pollution monitoring/measurement 659 Biotechnological methods for measurement ‘ot pollution 659 Citeria for biomonitoring of pollution 659 Bioassays in environmental monitoring 660 Cell biology in environmental monitoring 661 Molecular biology in environmental monitoring 682 Biosensors in environmental monitoring 653 Biotechnological methods for management of pollution 663 ‘Atmospheric CO, reduction 668 Conran Dera, Photosynthesis to reduce atmospheric CO, 664 Biological calcification t0 reduce atmaspheric CO, Sewage treatment by bacteria and algae Eutrophication and phosphorus pollution Biological removal of phosphorus Management of metal pollution Bioscavengers of metals ‘Mechanism of metal scavenging Immobilized cells in the management of pollution 55 Air Pollution and Control Sources of air pollutants Classification of air polltanss Feats of air pollution Biomonitoring of air pollution Air polltion control Control devices ior particulate pollutants Control devices for gaseous pollutants Control devices for volatile oanic ‘compounds (VOCS) 56 Water Pollution and Sewage Dissolved oxygen Water pollution ‘Nature of water polltans Onzanic pollutants Inorganic pollutants Microbiological pollutants Radioactive pollutants Waste water and sewage Composition of sewage Types of sewage Measurement of water pollution “Measurement of organic matter of sewage Detection of pathogenic organisms of senage Laboratory methods for detection of coliform organisms Techniques to distinguish fecal fom norefecal bacteria 57 Sewage/Waste Water Treatment Classcaion of treatment processes 664 658 665; 666 666 656 666 667 669 669 670 on or or 673 675 678 679)__ CONTENTS IN DETAIL Preliminary treatment Primary treatment ‘Secondary oF biological treatment Aerobie suspended-growth treatment processes Acivated sludge process ‘Acted lagoons. Senuencing batch reactor Aerobic digestion Aerobic attached-growth treatment processes “Tickling fiers Roughing fiers Rotating biological contactors (RBC) Packed-bed reactors iuitized bed reactors) Anaerobic suspended growth treatment processes Anaerobic digestion Anaerobic contact process Anaerobic attached-growth treatment processes Anaerobic iter process Expanded-bed process Pond treatment processes ‘Aerobic ponds ‘Anaerobic ponds Facultative ponds Tertiary treatment Solids removal Biological nitrogen removal Biological phosphorus removal Disinfection Sewage/vaste water treetment — a summary Waste water treatment of some industries ‘Waste watee eaten for eaiies Waste water treatment for distillery Waste water teatment for tannery Waste water treatment for sugar industry Treatment of antibiotics in waste water Water recycling Small sewage/waste water treatment ystems ‘Onidation ditch Septic tanks Ibo tanks 687 688 689 690 590 631 at 692 ox 62 693 693 693 cat 695 696 696 696 696 696 696 697 697 698 699 700 701 703 703 703 7 708 708 705 705 705 706 706 706 707 58 Sludge and Solid Wastes-Treatment and Disposal 708 Sources and characteristics of sludge 708 Preliminary operations 708 Suge thickening (concentration) 709 Sludge stabilization 70 Composting m ‘Mechanism of composing 72 Methods of composting m2 Vermicomposting m4 Conditioning of sludge 74 Disinfection of sludge 74 Dewatering 7™4 Heat drying ns ‘Thermal reduction of sludge ns Ultimate disposal of sludge 75 Landing 78 Lagooning 716 Seprage and sepiage disposal 716 Treatment and disposal of solid wastes n6 Separation and composting plans nz ‘59 Biodegradation and Bioremediation 718 Pseudomonas —the predominant ‘microorganism for bioremediation 718 Recalcivant xenobiotics 720 “Types of bioremediation 720 Metabolic effects of microorganisms on xenobiotics ma Types of reactions in bioremediation m2 Biedegradation of hydrocarbons m Biodegradation of pesticides and herbicides m Biodegradation of some other important compounds na Genetic engineering for more efficient bioremediation na Genetically engineered microorganisms (GEM in bioremediation 726 Bioremediation of contaminated soils and waste lands nz “Techniques of sll bioremediation 77 Bioremediation of ground water 860 Global Environmental Problems Green house efeet and global warming Green house gases Measures to contol green house effect The problem of ozone Depletion of ozone (Ozone hole fects of ozone depletion ‘Measures to control ozone depletion ‘Acid rain Development of acid rain Effets of acd rain ‘Measures to contol acid rain Environmental sustainability and biotechnology Biotechnology and Society 61 Biotechnology — Society, Risk, Ethics, Patenting Benes of bioteehnolegy ELS! of biotechnology Recombinant Hrapeutc product for human healthcare Genetic mifiations an food consumption Recombinant foods and religious belias 742 ‘re GM ods safe 742 Release of genetically engineered organisms. 742 ‘Applications of human genetic rDNA research 743 Human embryonic stem cell research 744 ‘Cloning humans? 744 Patenting biotechnology inventions 744 Biotechnology products 744 Biotechnology processes 744 What i a patent? 744 Iellecuat property its 744 ‘The process of patenting 745 Plant breeders hts 746 Patenting and biotechnology research 746 Biotechnology and the developing counties 746 Convers me Dena Basies to Learn Biotechnology 62 Cell—The Basic Unit Life Prokaryotic and eukaryotic cells Eukaryotic cell ‘Anima cell Plant cell Gxt Characteristics of bacteria Imporance of bacteria Control of pathogenic bacteria Fungi Characteristics of fungi Imporance of fungi Control of fung Viruses Characteristics of viruses Biological satus of viruses Importance of viruses ‘64 Bioorganic and Biophysical ‘Chemistry, and Biochemistry Tools Introduction to bioorganic chemistry Most common organic compounds found in living system Common functional groups in biochemistry Common ring structures in biochemistry ‘Acids and bases ters Solutions Colloidal state Diffusion Osmosis Viscosity Surface tension Isotopesou CONTENTS IN DETAIL Tools of Biochemistry 766 Enzyme inhibition 780 Chromatography 768 Enzyme specifiy 71 Electroporesis 767 Goenzymes 792 Phoiometry —colorimer and Mechanism of enzyme action 793 Specrophotometer 767 Diagnostic imporance of enzymes 795 Ulvaceniusation 768 oenaymes 795 Radioimmunoassay 769 ‘ Enzyme-linked immunosorbant assay 770-67 Metabolisms 796 Introduction to metabolism 736 65 Biomolecules 771 Victabolien'el esto byeat 738 Chemical molecules of tie 771 Ghyolsis 798 Carbohydrates 772 Conversion of acetyl CoA to pynnate 798 Classification of cabobydates 772 Cite acid eyle 799 Monosaccharides 773 Gluconeagenesis 800 Giycosies 773 Glycogen metabolism 800 Derivatives of monosaccharides 773 Glycogeness 200 Disacchardes 774 Glycogenoysis 200 Polysaccharides 724 Hexose monophosphate shunt 200 Homopolysacchardes 774 Giyoryate cycle 03 Heteropolsaccharides 773 Photosynthesis 803 Lipids 775 Metabolism of lipids 04 Clasiieation of pis 725 Faty acid oxidation os Faty acids 776 Biosynthesis of faty acids 806 “Wacyshyceols 776 Metabolism of cholesterol 206 Phospholipids 776 Cholesterol biosynthesis 206 Lipooroeins 777 Desadaton of cholestrot 206 Steroide 777. Metabolism of Amino acids 806 Proteins and amino aci 777 Metabolism of amin acids — ‘Amino acids 778 general aspects 208 ret rviee 761 Tansamination 809 Primary stuctire of protein 781 Deamination 09 Secondary structure of protein 782 Urea cycle 809 Tertiary strvture of protein 783 Metabolism of individual amin acids 009 Quaternary suture of protein 783 Glycine 809 sscation of proteins 783, Phenylalanine and tyrosine 209 Ihoprenoids and plgments 784 Typrophan a ee 785 Sulfve amino acids ait Es 705 Glutamate and glutamine sm Fate of carbon skeleton of amino acids it 66 Enzymology 786 Integration of metabolism 812 Nomenclature and classification 786 Energy demand and supply an ‘Chemical nature of encymes 787 Integration of majoe metabolic Factors afiecting enzyme activity 787 pathways and energy metabolism 813 atv site 789 Regulation of metalic pathways ais ee68: Immunology trate mit The immune system Onganzation of immune system Cel ofthe immune system Immnrlebins Smut of mmunoslbulins Clases of immunoglobulins Synthesis of immunoglobulins Major hstcompatibilty complex The immune response ytoknes Immunity in helth and disease ‘Autoimmune diseases Organ transplantation Cancers ‘NDS 69 Genetics Brief history and development of genetics Basic principles of heredity in humans Patterns of inhertance Genetic diseases in humans Eugenics 10 Bioinformatics Broad coverage of bioinformatics ‘Components of bioinformatics Bioinformatics and the internet Biological databases Applications of bioinformatics Appendices Glossary Abbreviations IndexDD tte chemical vehicle of here, is ‘composed of functional units, namely genes ‘The term genome refers tothe total genetic infor mation contained in a cell, The bacterium Escher: cha calf contains about 4400 genes present on @ Single chromosome, The genome af humane ic ‘more complex, with 23 pats of (diploid chromo- somes containing 6 billion (6 x 10%) base pairs of DNA, with an estimated 30,000-40,000 genes. At any given time, only a faction of the genome Is expressed ‘The living cells possss a remarkable property to adapt to changes inthe environment by regulating the gene expression. For instance, insulin. is synthesized by specialized cells of pancreas and rot by cells of other organs (say kidney, liven, although the nuclei of all the cells of the body ‘contain the insulin genes,” Malecular regulatory ‘mechanisms facilitate the expeession of insulin gene in pancreas, while preventing its expression in cher cells GENE REGULATION—GENERAL The regulation of the expression of genes Is absolutely essential forthe growth, development, Uifferentiation and the very existence ‘of an ‘organism. There are two types of gene resulation- positive and negative, 1. Positive regulation : The gene regulation Is said to be postive hen its expression is increased by a regulatory element (postive regulator Se Seen asia ST AE aie eg loans NA SOR 2. Negative regulation : A decrease inthe gene ‘expression due 10 the presence of regulatory clement (negative regulator) Is referred 10. as negative regulation Ik may be noted here that double negative effect ‘on gene regulation rests in a positive phenomenon. Constitutive and inducible genes ‘The genes are generally considered under two categories, 1. Constitutive genes : The products (grates) cof these genes are required all the time in a cell Therefore, the consitutve genes (or housekeeping genes) are expressed at more or ess constant rate in lost ll he cells and, further, hey are nat subjected 10 mauaton eg. the enzymes of cite acid cycle, 2. Inducible genes : The concentration of the proteins symthesized by inductble genes regulated by various molecular signals, An inducer increases the rxpeossinn of these gene while a pressor decreases, eg uyptophan pyolase of liver is Induced by tryptophan. One cistron-one subunit concept ‘The chemical product of a gene expression is a protein which may be an enzyme. it was orginally believed that each gene codes for a specific enzyme, leading to the popular concept, one gene- fone enzyme This however, i-aot necessarily valid ‘due to the fact that several enzymes (or proteins)are composed of two or more nonientical subunits {olypeptie chains). ‘The cistron is the smallest unit of genetic ‘expression. It isthe fragment of DNA coding forthe subunit of 2 protein molecule. The original concept of ane geneone enzyme is replaced by one Elucidation of the regulation of gene expression in prokaryotes has largely helped to understand the Brinciples ofthe low of information from genes to mRNA to. synthesize specific proteins. Some Important features of prokaryotic gene expression are described fist, Ths is followed by a brief account of eukaryotic gene expression The operon is the coordinated unit of genetic ‘expression in bacteria. The concept of operon was Introduced by Jacob and Monod in 1961 (Nobel Prize 1965), based on their observations on the regulation of lactose metabolism in Eco This popularly known as lac operon. LACTOSE (LAC) OPERON Structure of lac operon ‘The lac operon (Fig 5.1) consists of a regulatory zene (; | for inhibition), operator gene (O) and three structural genes (Z, Yl. Besides these genes, there is a promoter ste (F), next to the operator gene, where the enzyme RNA polymerase binds, The structural ones Z. Y and A respectively, code for he enzymes “Sesaacositas—slcente permease and galac B-Galacto. BIOTECHNOLOGY Repression of lac operon The regulatory gene (Dis consutive, I i ‘expressed ata constant ate leading tothe syathesis ot ae repressor lac pressor a tetrameric @ Subunits) regulatory protein otal mol. wt 150,000) ‘which specially binds tothe operator gene 0), This prevents the binding of the enzyme RNA polymerase to. the promoter site (P thereby blocking the tanscrigton of structural genes 2, ¥ and A) This i what happens in-the absence of cin The reper mole aca negative regulator of gene expresion, Dorepression of lac operon In the presence of lactose (inducer) in the medium, a small amount of it can enter the E. coi cells. The repressor molecules have a high afinity for lactose. The lactose molecules bind and induce conformational change in the repressor. The res. {is thatthe repressor gets inactivated and, therefore, cannot bind to the operator gene (O}- The RNA, polymerase attaches to the DNA at the promoter Site and wanserition proceeds, leading, tothe formation of polyestronic mRNA (for genes Z, ¥ and A) and, finaly, the 3 enzymes. Thus, lactose Induces the synthesis of the three enzymes Bezalactosidase, —galactosde permease and salactoside accilase. Lactose acts by inactivating the repressor molecules, hence this process is knowa as derepression of lac operon Gratuitous inducers: There are certain structural analogs of lactose which can induce the lac operon but are ot the substrates for the enzyme Be salactosidase. Such substances are known 36 _rauitous inducers, bopropythiogalactoside (FTG) is a gratuitous inducer, extensively used for the study of lac operon, “The eatahaite gone activator protsin: The calle of Ecol utilize glucase in preference to lactose; $iatehycrofses icone basset to galcione when bth of them are preen the medium, and lacose while Bernese i sponse fr he Iransport of lactose ito the cell The function of acetylase ins a mystery “The stuctual genes Z, Y and A wanscibe into single large mRNA with 3 independent translation nits for the synthesis of 3 lsinet enzymes. An TaRNA coding for more than one protein = known 5 poiycistronie_mRNA. Prokaryatic organisms contin # large number of poycistonic mRNAs. ‘Ater the depletion of glucose in the medium, utlizato of lactose starts. This indicates that lucose somehow interferes withthe induction of lac operon, Ths i explained as follows. The attachment of RNA polymerase to promoter ste requires the presence of a {gene activator protein (CAP) bound 1 cyclic (fig. 5.2). The presence of glucose lowers inracelular concentation of cAMP by inactvai(Chapter 5 : REGULATION OF GENE EXPRESSION Operator megan hae ose = aes Posi[poa|e 25 He as Ba) tee nea feat aE | t Aa au Edo ae a oo Erp rea ce av | @ | ES ey Sr erage OO. jet oo EA iy t: fee ie lel Inactive PGolaciosidase Pemnease Aceiyiase ‘Fg. 64 : Medel o acoso operon in Ecol (A) Stucure of lc apern (B) Repiesion ofl operon (CAP—catar—cataotte (©) Derspression cf ize peran. ‘ne acvater roe Bound Yo AMP: ANAP-ANA poiymaraes) the enzyme adenyiyl cyclase responsible forthe synthesis of cAMP. Due to the diminished levels of AMP, the formation of CAP-CAMP ison. Therefore, the binding of RNA palymerase to ONA (due to the absence of CAP-cAMP) and the transcription i almost negligible in the presence of lucose, Thus, glucose interferes with the expression of lac operon by depleting cAMP evel ‘Adéition of exogenous cAMP is found to initiate the transcription of many inducible operons, Including lac operon, Ics now clear thatthe presence of CAP-cAMP is esental for the transcription of structural genes OF lac operon. Thus, CAP-cAMP acts as a postivecoe yoo Bestig Giicose te ip 6 x0 SOOMEIEBEE yey ow. a car cae (2) Pore ered al Pavyitone mANA ia. 52 + Conte of ao pron by catabolite gone {actvalor poten (CAP) andthe rae of gucase, regulator for the gene expression. Iti, therefore, tevident that lac operon is subjected to both postive {by repressor, described above) and negative regulation, “Typtophan is an aromatic amino acid, and is requited forthe synthesis of all peoteins that contain Inptophan. I tryptophan is not present in the ‘medium in adequate quantity, the bacterial cell has to make it, as itis required forthe growth of the bacteria, ‘The typhan operon of Ecol s depicted in Fig, 5.3. This operon contains five structural genes Ape, "pD, tr2C, 18, tpA\, and the regulatory lement-—primary promoter (pM, operator (PO), attenuator (pa, secondary. internal promoter (pp), apd terminator (up “The five stuctural genes of eyplophan operon code fr thee enzymes {two enzymes contain vo diferent subunits) required for the synthesis of Luyptophan from chlorismate. “The tryptophan represtor is always turned on, unless itis represed by a specific molecule called BIOTECHNOLOGY corepressor, Thus lactose operon (described sltealy) is inducible, wheceas tryptophan operon is ‘repressible. The tryptophan operon is said to be Gepressed when it fs actively transcribed. “Typiopla is as » corepressor to hut down the synthesis of enzymes from tryptophan operon. This fs brought oUt in association with a specific protein, namely teyptophan repressor. Typtoohan repressor a homodimer (contains two identical Subunits binds with two molecules of tryptophan, land then binds to the tp operator to turn off the ‘tanscription. It of interes to note that tryptophan repressor also regulates the transcription of the ene (upR} responsible fr its own synthesis “wo polycistanie mRNAS are produced from tryplophan operon—one derived from all the five Structural genes, and the other obtained from the last three genes. Besides acting as 2 corepressor to. regulate leyptophan operon, teyptophan can inhibit the activity ofthe enzyme antvanilate symbetase. This is referred to as feedback inhibition, and is brought cut by binding of ryptophan at an allosteric site on anihrailate synthetase, ‘Attenuator as the second control site for tryptophan operon Axtenuator gene (vp) of tryptophan operon lies ‘upstream of t€ gene. Attenuation isthe second level. of regulation of tryptophan operon. The attenuator region provides RNA polymerase which Tegulates tanscription, In the presence of tryptophan, transcription is prematurely terminated fl the end of altlenuator region. However, in the absence of tryptophan, the atenuator region has ro ‘efiect on transcription. therefore, the polycsironic MRNA of the five strucural genes can be synthesized ach cell of the higher organism contains the entire genome. As in prokaryotes, gene expression in eukaryotes is regulated (0 provide theGuiamine PREP appropriate response to biological needs. This may cur in the following ways + Expression of certain genes (housekeeping genes) in most of the cells, + Activation of selected genes upon demand. + Permanent inssivation oF several genes in all but 2 few types In case of prokaryotic cells, most of the DNA ‘organized into genes which can be transctbed. In ‘contrast, in mammals, very litle of the total DNA is organized into genes and their associated regulatory sequences. The function of the bull: of the extra DNA is not known, Eukaryotic gene expression and ts regulation are highly complex. Some of the important aspects ‘are briefly described 5 = REGULATION OF GENE EXPRESSION 1 ‘STRUCTURAL GENES: REGULATORY ‘pt ELEMENTS, | | mRNAs peo | eh Pet etme ee ete, ee Fg “emeaet pte, Jette pecroes fransterase indole glycerol pte esr ee ois an 7 covrtsxes ee ee ie 1 aniandate ne ICES FE ACTONS string Fig. 6.3. Typtophan opsran in Ecol (regulatory elements are promoter (ipP), operator (pO), attenuate (tea) secondary intemal promote (rpPs) nd teminator (4p); Col, Col Cameenent J and : ‘-aephosphate: CARP — Carboxyohenylamine ul CHROMATIN SRUCTURE AND GENE EXPRESSION The DNA jin higher organisms is extensively folded anc packed to form protein-DNA complex called chromatin. The structural organization of DNA in the form of chromatin plays an important ole in. eukaryotic gene expression. In. fact, chromatin structure provides an additional level of control of gene expression [A selected list of genes (represented by the products) along with the respective chromosomes fn which they are focated is given in Table 5.1. In.general, the genes that are transcribed within ® particular cell are less condensed and more open in structure. This is in contrast to genes that ave not transcribed which form highly condensed chromatina] list of genes (represented by the products) along with respective chromosomes Genes ‘Chromosome ‘number ‘Akale phospalase 1 ‘Alpopoien B a TTransferin 3 ‘Nootol dehyrogenase 4 HUG CoA reductase 5 Stra 2-ycronyase 5 Aginasa Z Carbonic antytrase a Irertren ° Paathyrid hormone 1 yceradehyce Sphosphale dehydrogenase 12 Aderosine deaminase 13 ca Aitypsin 4 ‘ojochrone Pay 5 Hemogicincechin 6 Growth horene 7 Preabumin 18 Creatine phosphokinase (M ch) 8 ‘Aderosnedearinaso 2 ‘Siperxide druase a Immunoglobuin (cain) 2 (Gucnee 6 phosphate dehydrogenase x Stood eutetase % Histone acetylation and deacetylation Eukaryotic DNA segments are wrapped around histone proteins 10 form nucleosome. Acetylation ‘or deacetylation of histones is an important factor in determining the gene expression. in general, Acetylation of histones leads to activation of gene ‘expression while deacetylation reverses the effect. Acetylation predominantly occurs on the lysine tesidues in the amino terminal ends of histones ‘This modification in histones reduces the positive ‘charges of terminal ends (tails), and decreases thelr binding affinity 10 negatively charged DNA. Consequently, nucleosome siucture is disrupted to allow trancrition, BIOTECHN Methylation of DNA and inactivation of genes Cytosine in the sequence CG of DNA methylated. to form 5'-methyleytosine.. portion of CG sequences ‘about 20%) in ul DNA exists in methylated form, In ger ‘methylation leads to los of transcriptional act and thus inactivation af genes. This occurs due binding of methylcytasine binding proteins methylated DNA. As a result, methylated INA ‘ot exposed and bound to transcription factors, Interesting to note that methylation of DI Correlates with deacetylation of histones Provides a double means for repression of The activation and normal exprestion of gt and_gene inactivation by DNA methylation depicted in Fig. 54 oe Transeripion stare Srey so (e ‘Gone activation ‘and expression o onset Gy CHa Cy = Gene inactivation and no ex Fig. 8.4 : Methylation of DNA and genes (A) Gono activation in the absence mettylation (B) Gane inactivation due fo ___Q represent og [eee(Chapter 5 : REGULATION OF GENE EXPRESSION ENHANCERS AND TISSUE-SPECIFIC GENE EXPRESSION Enhancers (or activators) are DNA elements that facilitate or enhance gene expression. The enhancers provide binding sites for specific proteins that regulate transcription. They facilitate binding ‘of the transcription complex to promoter regions. Enhancers differ from promoters in two distinct ways 1. Enhancers may be located thousands of base pairs away from the start of transctiption site (promoters are close to the site of tanseription). 2. They can work in either orientation i.e. tenhancers can work upsteam (5) or downstream {31 from the promoter Several eukaryotic genes containing enhancer elements at various locations relative to their coding regions have been identified Some of the enhancers possess the ability to promote transcription in a tissue-specific manner. For instance, gene expression in lymphoid cells for the production immunoglobulin (i) is promoted by the enhancer associated with ig genes between J and C regions. Transgenic animals are frequently used for the study of tissue-specific expression, The available evidence from various studies indicates that the tissue-specific gene expression is largely mediated through the involvement of enhancers. COMBINATION OF DNA ELEMENTS AND PROTEINS IN GENE EXPRESSION Gene expression in mammals is @ complicated with several envieonmental stimuli ona le gene. The ultimate response of the gene ich may be positive oF negative i herwght ev the association of DNA elements and protein. Inthe illustration given in the Fig. 5.5, gene Lis by a combination of activators 1, 2 and 3, Nl is more effectively activated by the ined action of 1, 3 and 4. Activator 4 is not t contact with DNA, but it forms a bridge activators 1 and 3, and activates gene il is gene il, it get inactivated by a jon of 1, 5 and 3. In this case, protein 5 With the binding of protein 2 with the activates the gene. 8 L Gene naetvates Fig, £5 : A degrammatc represeniation of the ‘association of DNA elomonts and proteins in gene regulation, A,B and G represent gnes I 1! and (1.5 roproeant protein) MOTIFS IN PROTEINS AND GENE EXPRESSION ‘A. motif Iiterally means a dominant element Certain motifs in proteins mediate the binding of regulatory proteins (transcription factors) to DNA. The specific contol of transcription occurs by the binding of regulatory preteins with high affinity to the corect regions of ONA, ‘A great majority of specific protein-DNA {interactions are brought out by four unique moti ‘+ Helix-turn-helix (HTH) # Zine finger * Leucine zipper «+ Heli-loop-helix (HLH. “The above listed amino acid motifs bind with high affinity tthe specific ste and low affinity to ther parts of DNA. The motiADNA interactions are ‘maintained by hycrogen bonds and van det Waals forces, Helixturn-helix moti The helicturn-helix (ATS) motif is about 20 amino acids which represents 2 small part of a lange protein. HTS i the domain part of the proteinwhich specifically interacts with the DNA (ig 5.64). Examples of helisturn-helic motif proteins include lactose repressor, and cyclic AMP Patabolite activator protein (CAP) of E.coli, and Stveral_ developmentally Important. transcription For in mammals, collectively vefered to 36 hpomeodomain proteins, TRe term homeodomain Teles, 10 the portion of the protein of the anecription factors that reuuynizes DNA. Hameodomain proteins play a Key role in the development of mammals. Zine finger motif Sometime age, it was recognized that the transcription factor TFIIA requires zinc for its Seti On analyst twas Tevestee that each TRIIA contains zinc ions as a” repeating coordinated complex. This complex is formed. by fhe closely spaced aminoacids cysteine and steine followed by a histidine histidine pat In Sine instances, His-His is replaced by a second Cye-Cys pale (Fig. 5.681, “The zine fingers bind to the major groove of [DNA and lie on the face of the DNA. This binding rakes a contact with > bq of DNA, The sterol Thormone receptor transcription factors use 2iN Finger mati to bind to DNA. ‘The occurrence’ of a mutation resulting in @ single amino acid change of zinc finger may lead to existance to the action of certain hormones on gene expression. A mutated zinc finger resistant © the action of calctil (active form of vitamin D) thos been identified. This may ultimately result #9 rickets (vitamin D deficiency Leucine zipper motif ‘The basic regions of leucine zipper (bZIP) proteins are rich isthe amino acid leucine, There Pccury a peviodic repeat of leucine residues =t every seventh position. This type of repeat structure Sows two identical monomers or heterosis t Zip together and frm a dimeric complex. This Groteneprotein complex associates and interacts fith DNA (Fig. 5.60). Good examples of levcine “Tipper proteins are the enhancer binding proteins (EBP!—fos and jun. Helixcloop-helix motit “wo amphipathic (iterlly means a feeling of closeness) achelical segments of proteins can form fralicloophelix motif and bind to DNA. The (8) Neti tenho (©) Leucine zipperimportant features of eukaryotic gene on along with the regulatory aspects are in the preceeding pages. Besides tion, eukaryotic cells also employ variety mechanisms to regulate gene expression ost Important ones are listed below, and described next i. Gene amplification Gene rearrangement Processing of RNA, Alternate mRNA splicing “Tansport of mRNA from nucleus to cytoplasm Degradation of mRNA. amplification this mechanism, the expression of a gene is < Several fold. Ths is commonly observed the developmental stages of eukaryotic . For instance, in fruit fly (Drosophila), plication of genes coding for egg shell is observed during the course of oogenesis. plication of the gene (DNA) can be lunder electron microscope (Fig. 5.7 occurrence of gene amplification has also ted in humans. Methotrexate isan ‘drug which inhibits the enzyme plate reductase. The malignant cells drug resistance to long tezm administration exate by amplifying the genes coding for body possesses an enormous capacity to ze a wide range of antibodies. It is ‘thatthe human body can produce about (109) antibodies in response to antigen The molecular mechanism of this ity Was not understood for Jong. It is Se Fig. 57 : Diagrammatic represeniation ot ‘now explained on the basis of gene rearrangement 0 transposition of genes or somatic recombination of DNA ‘The structure of atypical immunoglobulin mole. cule consists of two light (L) and two heavy (H) chains, Each one of these chains (L or H) contains an N-terminal variable (V) and C-terminal constant {© regions, The V regions of immunoglobulins are fesponsible for the recognition of antigens. The Phenomenon of gene rearrangement canbe understood from the mechanism of the synthesis of light chains of immunoglobulins (Fig. 5.8 Each light chain can be synthesized by three distinct DNA segments, namely the variable (V), the joining ) and the constant (,). The mamma: lian genome contains about 500 Vj segments, 6 ), segments and 20 C, segments. During the course af differentiation of Bslymphocytes, ane V, segment (out of the 500) is brought closer to. and Cy Segments, Ths occurs on the same chromosome For the sake of illustration, 100" V,, 34), and 10° C,_ segments are rearanged in” Fig. 5.8. The rearranged DNA (with Vi, J, and C, fagmen) is then transcribed to produce a single mRNA for the synthesis of a specific light chain of the antibod, By innumerable combinations of V,, h and Cy segments, the bodys immune sytem can generate millions of antigen specific immunoglobulin molecules. The formation of heavy (H) chains of immuno- elobulins also occurs by.rearrangement of 4 distinct -genes—variable (V,), diversity (D), joining Uy) and Constant (C,qBIOTECHNOLOGY (600) 10 ce Sy pirate $} Ofna ONA ae ~ Rearenged DNA Vee | btm SH Pemary ransctot ae ae ey a tip cha tt) “Fig 5: Bagranmatereprconiaion of 300 re ayes fit can of immunegebuh. Processing of RNA The RNA synthesized in transcription undergoes ‘modifications resting in a funcional RNA. The ‘chants include itron-exon splicing, polyadeny tion ete (Chapter 4) Alternate mRNA splicing Eukaryotic cells are capable of carrying out altemate mRNA processing to control gene ‘expression. Different mRNAs can be produced by alternate splicing which code for diferent proteins for more details, Refer Chapter 4), Degradation of mRNA ‘The expression of genes is indirectly influenced by the stability of mRNA. Certain. hormones regulate the synthesis and degradation of some mRNAs, For instance, estradiol prolongs the half- life of vitellogenin mRNA from a few hours t0 sbout 200 hour cap s-Nos] Coding agin |s-NOS}-A-A~A), [Bee a anos Reiac.saen representation of atypical te appears that the ends of mRNA molecules etermine the stability of mRNA. A typical teukaryatic mRNA has S-non-coding_ sequences (GENCS), a coding region and 2 3'NCS. All the TIRNAS are capped athe en, al est of Ha hhave a polyadenylate sequence at the 3” end (Fig, 5.9. The 5’ cap and poly (A) tal protect the ‘RNA against the attack by excnuclease. Further, stermogp structures in NCS regions, and AU fich regions in the 3” NCS also provide ably to mRNA. Gene expression of gene regulation is usually studied atthe vanscriptional level, ie. production ‘of mRNA from the gene. The methods to elucidate gene expression are designed to provide information ‘08 one oF more ofthe fllowing += Sequence of the gene + Size ofthe transcript (mRNA) ‘+ Starting and finishing points of genes to produce the transerit ‘+ Number and position of introns on the genes. +The activity ofthe promoter Some of the important and general methods employed to study the gene regulation are brily described,5 = REGULATION OF GENE EXPRESSION orn blot Southern blot is a novel technique to detect a fragment of DNA in the DNA preparation of ‘organism. Ths technique is paticulaey useful 10 the presence of a foreign DNA in the ally modified organisms oF to identify the esence and copy number of genes in an sniam’s genome. The details on this technique them blot Norther blot specifically detects the size and quence of the MRNA. The total mRNA is facted from a cell or tissue suspension, and Separated by agarose gel electrophoresis and then fetected by hybridization (Refer Chapter 7), eS mapping ‘Nuclease SI is an enzyme'that can specially ade single-stranded nucleic acids. Nuclease SI ing is used to_determine the number of prosent in a gone (Fig. 5-10. “The mature mRNA is hybridized with is corresponding gene (Le. genomic DNA). The ertion of the intron on the gene which is not scribed fs looped out. This looped-out intron be specifically digested by nuclease SI, which egrades the single-stranded DNA. The number ei presence of introns can be identified by Jysing the fragmented DNAS. lease protection assay In nuclease protection assay, she test ranscript RNA) is hybridized with excess quantities of in @ synthesized and radioactively labeled DNA plecules (usually obtained fom cloned genes) annealed hybrids which are labeled, are jected © digestion by nuclease SI which grades single-stranded nucleic acids. The eaweineated end uuteated —liyLaidized plecules are separated by agar gl electrophoresis didnt (Fig. 5.1. Nuclease protection assay is a vatiant of ease SI mapping and provides information as ds the presence of intons, transcriptional ini and the test transcript proper. extension Primer extension method is arellable technique ‘> ine the 5° end of the transcripts For this 2 synthetic 5labeledoligonucletide primer Eco Iron xen _Inron_Exon 25 mucease si ios | mRNA Tabaled BNA | “Tost tanec (FINA) Asnesing | | uctase si fo Batik ~Nueleste protected ‘agent And B subjecod io lectoporesis and ‘suraaography1 So c= "e eee om : ‘a : ei Poe = en Seceaniee Fla 6.12 +A dagranmatcrapresonaton of primer ‘ertension fecnique To detect the See age siege containing complementary base sequence toa small portion ofthe test transcripts used. Both are allowed to ibridize, and the enzyme reverse transcriptase tke to extend the primer til it reaches the 5” end of the mRNA (Fig. 5.22) ns resus In the syne of Complementary DNA (CDNA) representing the Gistance between 32nd of the primer and 5end of the mRNA. The cDNA can be separated by electrophoresis and detected. Rapid amplification of cDNA ends (RACE) The 5% and 3-ends of complementary DNA (CDNA) canbe mapped by use of polymerase chain Feaction. This method is eter 5-RACE or RACE ‘depending. on the end tobe mapped. For details on RACE, cefer Chapter 8. Reporter assays Reporter genes are the genes that form protein products whieh can be detected without Uesioying the tissuescells. To elucidate a gene expression its promoter fs fused with a reporter gene, and then introduced into the cel The specific products (ea, luciferase, rgalacto- sidase, chloramphenicol acetylvansterase) of the reporter genes can be identified, The activity ofthe Teporter gene reflects the activity of the promoter ‘gene, and consequently the gene expresion. Reporter asays are very useful forthe study of gene expression in vivo Inthe tsuestells. BIOTECHNOLOGY ‘Gene tagging broadly involves the insertion of recognizable DNA fragment within a gene so that the tinction of a gene is disrupted, and the gene is identifed by virtue ofthe inserted ONA fragment TEONA ttonsewred DNA) i the part nf tumor inducing plasmid (plasmid) DNA found inthe soi bacterium Agrobacterium tumefaciens (For detail Reler Chapter 49) Transposons or transposable ‘elements are mobile genetic elements (DNA pieces) that ean move fom dae place to another in a DNA ‘molecule (Refer Chapter 3. Trensposons or T-DNA, an be used in gene tagging and gene analysis “The transposon tagging ofa gene fs depicted in Fig, 5.13. When a transposon in a plasmid is inttoduced into a cel, it gets incorporated into DNA, and the gene gets disrupted. Consequently, transposon insertion produces a mutant (A. This ena nora ca) ee “Transposon 4 i [Fernotiton © a Gone adlonptes [maa To 7 moore ated ldeneaton of gone ‘Fiaggearanspcon Fig, 5:18: Tansposan tagging of gare inmutant can be identified by its phenotype and 2 ene library. Further, the mutant can be screened {or the presence of transposon. By identifying the Tocation of insertion of transposon, the location of the specific gene can be identified “The operation ofthe genome can be evaluated toy the study of prateome. Thus, by studying the functions of proteins, itis posible to understand how the genome operates and how a dysfunctional genome activity can result in disease slates such as Proteomics broadly invalves the methodolosy for characterizing the protein content ofthe cel, ‘This can be done by protein electrophoresis, mass spectrometry ec. Identification of protein-protein interaction is = recent approach to study proteome. The protein Interaction maps can be constructed to understand the relation between the proteome and cellular biachemisty. Phage display and yeast two-hybrid system are commonly used 1 study protein- ‘protein interactions PHAGE DISPLAY Phage display is @ novel technique to evaluate ‘genome activity with particular reference to identify Froteing that interact with one another. It basically Irvolves insertion of a foreign DNA into phage fenome, and is expresslon as fusion product with & eraae coat protein (Fig. 5.144). This is followed by Ctrening of tet protein by phage spay library {Fig 5.148, The technique is briefly described below. 'A special type of cloning vector such as 2 haceriophage or filamentous bacteriophage (e8 Mia) are used for phage display. A ragment of [BNA coding forthe test protein i inserted into the vector DNA fadiacent to phage coat protsin ene) Inter transformation of . coll, this recombinant ene (used frame of ONAT results inthe synesis BF hybrid protein, The new protein is made uP of the test protein used with the phage coat protein. ‘The phage patices produced in the transformed call display the test protein in their coats “The test protein interaction can be identified by sing a phage display library. For this purpse, the feat protein is inwmobiized within a well of @ Chapter 5 : REGULATION OF GENE EXPRESSION 7 ‘etor DNA T_ ane or phage i oat pon =o DNA tee proton say Insertion Fisadiame OWA Transformation of cok [es cD Pra etainoa phage “e¥4 : Euctaton of preter protein oration by phage depla (A) Proaucton of fusion proteih72 concosng Yat fa prey nroduced ete yeas el | Secon pasa BIOTECHNOLOGY Traneenptenal sewatonnain a eel ecaaras Fecombinant genes ey Cat | Sat Pry Teansetgiona actvatr Beng ‘one A Reporter gene |Transision epost prosin Fig, 15+ Eucdaton of potion ntracton by yeast wosyorid system (ANAP-ANA polymerase) ‘microtiter way, and the phage display library added. Alter several washes, the phages that are retained in the well are those displaying a protein that Interacts with the test protein, Phage-dspaying peptides canbe isolated, based fon their antibody-binding propesies, by employing afinty: chomatography. Several rounds of affiaity ‘Chromatography and phage propagation can be Used to ervich phages with desired proteins, Phagemid display Phagemid in place of plasmid, can also be used for the display of proteins. Infact, special tynes of [phagemid display vectors have been developed for this purpose. Phage and phagemid display can be successfully used for selecting and engineering. polypeptides With novel fonctions. YEAST TWO-HYBRID SYSTEM ‘Whee two proteins interact with each other, their corresponding genes are known as interacting tenes, The yeast two-hybrid system uses a reporter iene to detect the physical interaction ofa pair of. Proteins inside 2 yeast nucleus The two-hybrid method is based on the ‘observation that most ofthe transcriptional proteins {the proteins involved in promoting transcription ff a gene) contain two distinct domains — DNA binding domain and. transcriptional activation domain. When these two domains are pycally separated, the protein loses its activity. However, the same protein can be reactivated when the domains are brought together. These proteins can bind to! DNA and activate transcription, “The target protein is fused to a DNA-binding domain to form a bait. When this target protein binds to another specifically designed protein namely th prey inthe nucleus, they interact, which in tum switches on the expression of the reporter gene (Fig. 5.15). The reporter genes can be detected by growing the yeast on a selective medium Its possible to generate the bait and prey fusion proteins by standard recombinant DNA techniques. 2 single baid protein is frequently used to ish out eae feild antec h Mie Mette Ae prey proteins. large numberof prey proteins can be produced by ligating DNA encoding. the activation domain of a transcriptional activator toa misture of ONA fragments rom a CDNA library. Yeast three-hybrid system ‘The interactions between protein and RNA ‘molecules can be investigated by using a technique Known as yeast thre-hybvid system,on ae 6 Invoducion to Genetic ene 7 aie Techies in Cnet comm 2 8 Polymerase Cain Reaction "ONA Amptteston) 112 9 Game bates 120 10 Stedreced Mutagenesis and Patch Enewine 128 11. Maren of Gone Exresion | ines Cals, 137 12 Human Genome Projct_148tie engineering primarily involves: the pulation of genetic material (DNA) ‘0 the desired goal in a pre-determined way. ‘other terms are also in common use to genetic engineering ianipulation inant DNA (rDNA) technology cloning (molecular cloning) modifications genetics. HISTORY OF RECOMBINANT \ TECHNOLOGY the present day DNA technology has its roots in xperiments performed by Boyer and Cohen in In their experiments, they successiully mined two plasmids (pSC 101 and pSC 102), Cloned the new plasmid in Ecol. Plasmid pSC ossesses a gene resistant to antibiotic line while plasmid pSC 102 contains a gene At to another antibiotic kanamycin. The developed recombined plasmid when rated into the bacteria exhibited resistance eth the antibiotics-tetracyeline and kanamycin second set of experiments of Boyer and n were more organized. A gene encoding a in (required to form F@NA\ was isolated from Cells of African clawed frog Nenophs laevis, by ‘of a resviction endonuclease enzyme (ECORI, same enzyme was used to cut open plasmid pSC 101 DNA. Frog DNA fragments and plasmid DNA fragments were mixed, and pairing occurred between the complementary. base pals. By the addition ofthe enzyme DNA ligase, 4 recombined plasmid DNA was developed. These new plasmids, when introduced into Ecol, and grown on a rutrient medium resulted in the production of an extra protein i.e. the fog protein. Thus, the genes of a frog could be successiully transplanted, and expressed in Ecol. This made the eal beginning of rmadem *DNA technology and laid foundations for the present day molecular biotechnology. Some biotechnologits who admire Boyer CCohen experiments divide the subject into two Chronological categories. 1. BBC-biotechnology Before Boyer and Cohen. 2. ABChiotechnology After Boyer and Cahen. ‘More information onthe historical develonments of genetic engineering and biotechnology is given Under the scope of biotechnology (Chapter 1). ‘An outline of recombinant DNA technology “There are many diverse andl complex techniques involved in gene manipulation. However, the basic principles of recombinant DNA technology are reasonably simple, and broadly involve the following sages (Fig. 6.1. 1. Generation of DNA fragments and selection of the desired piece of DNA (eq, a human gene).76 ae Donor DNA fs oa nn vote en [alee "OS a we, O ar 2. Insertion of the selected DNA into a cloning Vector (e.g. a plasmid) to create a recombinant INA ur chimeric DNA (Chimera is 2 monster in Greek mythology that has a lio’s head, a goat's ‘body and a serpent’ tall, This may be comparable to Narasimha in Indian mythology) 3. Introduction of the recombinant vectors into host cells (eg. bacteria). 4. Multiplication and selection of clones containing the recombinant molecules. 5. Expression of the gene to produce the desired! product BIOTECHNOLOGY Recombinant DNA technology with special ‘eference to the following aspects is described in this chapter 1. Molecular tools of genetic engineering Host cells-the factories of cloning. Vectors-the cloning vehicles. Methods of gene transfer Gene cloning strategies, Genetic engineering guidelines. The future of genetic engineering, ‘An engineer isa petson who designs, constructs (24, bridges, canals, railways) and) manipulates according to a set plan. The term genetic engineer may be appropriate for an individual who. is involved in genetic manipulations. The genetic engineer's toolkit or molecular tools namely the enzymes most commonly used in recombinant DNA experiments are belly described RESTRICTION ENDONUCLEASES DNA CUTTING ENZYMES Restriction endonucleases are one of the most Important groups of enzymes for the manigulation of DNA. These are the bacterial enzymes that can ceuvaplit DNA (fom any source) at speclic sites ‘They were fist discovered in E.coli restcting the replication of bacteriophages, by cutting the vital DNA (The host Ecoli DNA is protected. from cleavage by addition of methyl groups). Thus, the {enzymes tha restrict the vial replication are known 8 restriction enzymes or restriction endonucleases Hundreds of rasvicion endonucleases have been isolated from bacteria, and some of them are commercially available, The progress and growth of ‘biotechnology is unimaginable without. the availability of resvicion enaymes. Nomenclature Restriction endonucleases are named by a standard procedure, with particular reference tothe bacteria from which they ae isolated. The fist letter (in italics) ofthe enzymes indicates the genusChapter 6 name, followed by the fist two lenors (also in italics) ofthe species, then comes the tain of the ‘organism and finally a Roman numeral inceating the order of discovery. A couple of examples are siven below. EcoRI is irom Escherichia (B coli (eo), stain RYI3 1 and frst endonsclones (Dt be dierowoeed Hindi is fom Haemophilus (H) influenzae (in, strain Rd (d) and the third endonucleases (HI to be discovered ‘Types of endonucleases ‘At least 4 diferent types of restriction endonucleases are known-type | (eg Ecok2), type (e.g, EcoR?, type Il (eg. OPN and type Is. Theit ‘characteristic features are given in Table 6.1. ‘Among these, type Il restriction endonucleases are ‘most commonly used in gene cloning. INTRODUCTION TO GENETIC ENGINEERING Tate 6.1 7 rises of ‘of restition endonucleases Type Salient features 7 Sige era wi Tar ‘expgin, clsavage ahd metyaon. Neen cleave up f 1900 bp tom recognition ste “wo oteronerzynes oir 1 leave oF oly he recogion Sequonce. Cleavage ste is he same or dose recogton ste, 1 le ena with 2 subs er i recognition a davage. Ceavage __Sit 2428 bp trom recognition ste, “no diferent enzymes, doavage sie is. up to 20 tp rom ecogniton sie t Enzyme (source) Recognition sequence Products com 5 RATT (Eschochi co 9 CTTAn 5) So GIG-AT-0-0-—2 FCT AGE Heel! s cdl (Haemophivs 2egyots) oC Hing 5 AG OTT 8! (aemophivs infuenzae) So TOG AYA 5! Wot! Sm GO BO08- 09 Ga0066. (Nocaraa omic) F-COOCEEES.5 eae eo oboe. (ote: Scsorsestetesesocavge Tha psu ar wih tans whe re ee pads ae wh ety nc| Recognition sequences Recognition sequence is the site where the DNA is cut by 2 restriction endonuclease Restriction endonucleases can specifically recognize DNA with a particular sequence of 4-8 hrucletides and cleave. Each recognition sequence has two fold rotational symmetry Le, the same uuketide sequence occurs on both strands of DNA which run in opposite direction (Table 6.2. Such sequences are tefered to as palindromes, since they read similar in bath directions (forwards and backwards) Cleavage patterns Majority of restriction endonucleases {particularly type I) cut DNA at defined sites within recognition sequence, A selected list of enzymes, recognition sequences, and their products formed is given in Table 6.2. The cut DNA fragments by _ restriction endonucleases may have mostly sticky ends [cohesive ends) o blunt ends, as gven in Table 6.2. DNA fragments with sticky ends are particularly tuseful for recombinant ONA experiments. This is because the single-stranded sticky DNA ends can easily pai with any other DNA fragment having complementary sticky ends DNA LIGASES—DNA JOINING ENZYMES The cut DNA fragments are covalently joined together by DNA ligases. These enzymes. were originally iolated from viruses. They also occur in Eccoli and eukaryotic cells. DNA ligases actively ppantcipated in cellular DNA repair process. ‘The action of DNA ligass is absolutely required fo permanently hold DNA pieces. This is so since the hydrogen honds formed between the ‘complementary bases (of DNA strands) are_not Song enough t© hold the strande together, DNA Tigase joins (seals) the DNA fragments by forming a phosphodiester bond between the phosphate sr0up fF 5-carbon of ene deoxysibase with the hydroxy! ‘group 3-catbon of another deoxyribose (Fig. 6.2) Phage T4 ONA ligase requires ATP as a cofactor while Ecali DNA ligase is dependent on NAD*. In teach case, the cofactor (ATP or NAD") is split to form an enzyme—AMP complex that brings about the formation of phosphodiester bond. The action ‘of DNA ligase isthe ultimate step in the formation ‘of a recombinant DNA molecule BIOTECHNOLOGY al elielede he E i Higa mol ens i roe $ we 8 8 GH, mas 5H, — ° ; one oe A i ; ? ape eee Homopolymer t ‘The complementary DNA strands can be joined together by annealing. This principle is wtlized in homopolymer tailing. The technique involves the addition of align (4A) tm Sends of some DNA molecules and the addition of liga (61) to 3-ends of other molecules. The homopolymer fextensions (by adding 10-40 residues) can be synthesized by using terminal deoxy- hucleotidyitransferase (of calf thymus. Homopolymer tailing, achieved by annealing is illustrated in Fig. 6.3. Linkers and adaptors _ Linkers and adaptors ae chemically synthesized, short, double-stranded DNA molecules. LinkersChapter 6 INTRODUCTION TO GENETIC ENGINEERING. possess restriction enayme cleavage sites. They can be ligated to blunt ends of any ONA molecule and fut with specific restriction enzymes t0 produce DNA fragment with sticky ends (Fig. 6.4. ‘Adaptors contain preformed sticky or cohesive ‘ends. They are useful to be ligated to DNA fragments with blunt ends. The DNA fragments held 10 linkers or adaptors are finally Ngated 2 vector DNA molecules (Fig 64) ALKALINE PHOSPHATASE. Alkaline phosphatase is an enzyme involved in the removal of phosphate groups. This enzyme is useful to prevent the unwanted ligation of DNA molecules which ia frequent problem encountered in cloning experiments. ‘When the linear vector plasmid DNA is treated with alkaline phosphatase, the 5-terminal Phosphate is removed (Fig 6.5). This prevents both ‘ecicularizaton and plasmid DNA dimer formation. itis now possible to insert the foreign DNA through the participation of DNA ligase. DNA MODIFYING ENZYMES Some authors prefer to use the broad tem DNA modiving enzymes to all the enzymes involved in fecombinant ONA technology. These enzymes represen the cutting and joining functions in DNA ‘manipulation. They are broadly categorized as frucleases, polymerases and enzymes modifying fends of DNA molecules, and briely described below, and ilsirated in Fig. 6.6. Nucleases Nucleases are the enzymes that break the phosphodiester bonds that hold sucleotdes together of DNA. Endonucteases 2c onthe internal ‘phosphodiester bonds while exonucTeases degrade DNA rom the terminal ends (Fig 6.68) Restriction endonucleases, desciTbed already, are good texamples of endonucleases. Some other examples fof enda- and exonucleases are listed. Endonucleases + Nuclease S, specifically acts, on single-stranded Dna ae leu ate + Deoxyribonuclease | [DNase I) cuts either single ‘or double-stranded ONA molecules at random Fig. 63. ES heed orsign DNA ae iearom Ea unkers os aie NA tease aa DNA with oil Foreign NA C= © Vector DNA Recombinant ONA "ip. 6 Use of inks (A) and aaa (8) the ‘oration of ecamanant ONA (ete atte kere ee chDNA as POH Plasmid eit pan Feciaarzaton tinaerD tla oe pn "= Se No easton HOH 4o——p 2 fo. Foreign DNA YONA isse Trager Fig, 65 + Acton of alkaline phosphatase to prevent the recieuarizatian(eigation) of vector plasmid, evek ao ‘Silesian ONAor ANA, ase ‘Double stranded DNA Double-standed DNA Double-stranded NA BIOTECHNOLOGY a OE came PF riemat ts Removal of nuclectises ee poe DNAterpiate RNA template = aa a Fig, 66 An outina of tho actutae of muionses (A), 4d DNA pobmerases (8). Exonucleases + Exonuclease | cuts DNA and generates molecules with protuding 5'-ends + Nuclease Bal 31 Is a fas acting 3-exonuclease. Its action is usually coupled with slow acting endonucleases. {A diagrammatic representation of the action of cendo-and exonucleases is given in Fig. 6.7. Besides the DNA cutting enzymes, there are NA specific nucleases, which are refed to as ribonucleases (BNases, Fig 67: Mote of action of solncod endo and exonsonses (DNase KDeonmbonuceaees Enzyme ‘Akane phosohaase alot nudeaee ONA igese NA puns | DNase | Exonuceate I > exonuclease Pouce kinase Festi enzymes Fevers tnscipase “The group of enzymes that catalyse the synthesis ‘of nucleic acid molecules are collectively referred to-as polymerases, It is customary to use the name ‘of the nucleic acid template on which the polymerase acts (Fig. 6.68}. The three important polymerases are siven below. “+ DNA-dependent DNA polymerase that copies DNA fram DNA, “+ RNA-dependent DNA polymerase (reverse transcriptase) that synthesizes DNA from RNA. 1+ DNA-dependent RNA polymerase that produces RNA from DNA. For more details on the functions of these enzymes, refer Chapter 4 Enzymes modifying the ends of DNA There are certain enzymes that act on the fexminal ends of DNA and modify these molecules. ‘The important ones are listed Allaline phosphatase that ermoves the terminal phosphate group (see Fig. 6.5, cleotide kinase involved in the addition of he most commonly used enzymes in recombinant DNA techaology/genetic ‘engineering Use/reaction | emaves phosphate groups tor Sends of dovbllsingl sanded DNA (or RNA) For tho progressive shortening of ONA. Jeins DNA melecis by forming prosghodtsterinkages between DNA segments Syuteszes DNA complementary toa DNA torpite. Produes single-stranded nicks in ONA. Femaves nucleotides tom 3rd of ONA. emaues nucleotides fom 5erd of DNA. * “Transl phosphate rom ATP to 8-OH ends of DNA or RNA. Cut ceute-stranded ONA wih a spade recopiton ste Syneszes DNA fom FINA Cleves and gusts the RNA stand of RNADNA heterodupien ‘dds rclatids tothe ends of DNA or ANA. Use in omopalymer ting ase A (leaves and digests RNA (an not DNA. Fase H Taq DNA polymerase Used in polymerase chain readon Si nuclease Degrades single standed ONA and RNA. Tern wasters Polymerases = Terminal transferase (also called terminal deoxynucleotidyl transferase) repeatedly adds ‘uceotides to any availabe 3'terminal ends, the ‘most suitable being the protruding 3ends, This enzyme is particularly useful to add homopolymer tails prior to the constuction of recombinant DNA molecules. ‘+ The most commonly used enzymes in recombinant DNA tachnologyigenetic enginearing ae listed in Table 6.3 The hosts are the fving systems or cell in which the cartier of recombinant DNA molecule or vector ‘ean be propagated. Tere are diferent types of host cellsprokaryotic (bacteria) and eukaryotic (fungi, animals and plans). Some examples of host cells Used in genetic engineering are given in Table 64 Host cells, besides effectively incorporating the vector’s genetic: material, must be conveniently Cultivated in the laboratory to collect the product Jn general, microorganisms are preferred 25 host cells, since they multiply faster compared to cells (of higher organism (plants or animals)2 PROKARYOTIC HOSTS Escherichia coli ey es eae tees cemenon eee aticeay ote rant seer Gatiey cook oe wee i sehen aaa ee ori toy ome Selo frome ay Sochecat Ue Se eeant Eel on Some rami See Tanne Tis, nba ly See eae CNA te Seve eo aml aati aatasrot eee er et omnes Pee te ee aoa ee i a Tedaora eg Ec aoe hws Corson a dares by ane tai omaion ee ee ieee einen oe opis Cacao coe meine ee a Initio. ecitus subs Bacills subtilis i @ tod shaped non-pathogenic bacterium. thas heen used as 2 host in incusty for the production of enzymes, antibiotics, insecticides fete. Some workers consider Bsubrilis as an alterative to Ecol EUKARYOTIC HosTS Eukaryotic ofganisms ate preferred to produce human proteins since these hosts with complex structure with distinct organelles) are more suitable fo synthesize complex proteins. The most commonly used euksryati. erganism isthe yeast Saccharomyces cerevisiae. tis 2 non-pathogenic organism routinely used in brewing and baking industry. Certain fungi have also been used in gene cloning experiments. ‘Mammalian cells, Despite the practical dificulies to work with and high cost factor, mammalian cells (uch as rouse cell) are also employed as hosts. The Advantage is that certain complex proteins which BIOTECHNOLOGY ‘mn genetic engineering Group Baamples Prokaryotle Baca chara call ‘aclu suis Sreptonyeae 6p Eurkaryotic Fung ‘Saccharomyces carvsian Asperlsniuons nina Insect cls eyes Maeraln cls Whol gaisms Plants Prooplasts it cals Wiole pais cannot be synthesize by bacteria can be produced by. mammalian cells eg. tissue plasminogen sctivetor This ie mainly becaure the: mammalian Cells possess the machinery to modiy the protein fo its final form (o0st‘ranslational mexiications) It may be noted here tha the gene manipulation ‘experiments in higher animals and plants are usually Caried out to alter the genetic make up of the fvgenism to create transgenic animals (Chapte'41) land transgenic plants (Chapter 4), rather than to isolate genes or producing specific proteins. Vectors are the DNA molecules, which can camry “a foreign DNA fragnient ta he cloned. They ate self feplicaing in an appropriate host cell. The most important vectors are plasmids, bacteriophages, cosmids and phasmids. Characteristics of an ideal vector ‘An ial vector should be small in size, with @ single restriction endonuclease site, an origin of feplicaion and 1-2 genetic markers (to identify recipient cells carrying vectors). Naturally occurring plasmid rarely possess all these characteristics.“hapten 6 : INTRODUCTION TO GENETIC ENGINEERING Plasmids are extrachromosomal, double fed, circular, seltrepicating DNA molecules. all the bacteria have plasmids containing a copy number (1-4 per cell) or 2 high number (10-100 per cell). The size of plasmids varies from 1 to 500 Kb. Usually ide contribute 0 about 0.5 to 5.0% af the DNA of bacteria (Note : A few bacteria linear plasmids eg, Steptomyces sp, la burger, of plasmids ere ave many ways of grouping plasmids are categorized as conjugative if they cary @ fof transfer genes (va genes) that facilites ial conjugation, and noa-conjugativ, if they ‘nat possess such genes. nother classification is based on the copy +. Stingent plasmids are present in limited (1-2 per cel) while relaxed plasmids occur number in each cel ide possess ganes fr their own tancor fone cell © another, while Replasmids cary resistance to antibiotic, seneal, the conjugative plasmids are large, Stringent contol of DNA replication, and are nt in low numbers. On the other hand, non- ative plasmids are small, show relaxed of DNA replication, and are present in high lature of plasmids 2 common practice 1 designate plasmid by case p, followed by the fist lets) of ss} names andthe numerical number given fe workers. Thus, pBR322_is a plasmid ed by Bolivar and Rodriguez who it as 322. ‘Some plasmids are ames ofthe places where they are discovered is plasmid from University of Calfoenia —the most common vector 322 of Ecol the most popular and widely plasmid vector and is appropriately regarded os oF grand parent of several other vectors, 122 has a DNA sequence of 4,361 bp. it genes resistance for ampicillin (Amp!) anc le ia Bam Sait gn of peaton Fig, 68 = Gonatc map of plasmid soning vector R22 tetracycline (Te) that serve as markers for the identification of clones carving. plasmids. The plasmid as unique recognition sits forthe action fof restiction endonucleases such a6 EcoR, Hind BamM, Sal and Pst (Fig. 6. Other plasmid cloning vectors ‘The ather plasmid employed as cloning vectors Include pUCtI9 2,685. bp, with ampicillin resistance’ gene), and derivatives. of pBR322~ pBR325, pBRIZ8 and pBRI29. BACTERIOPHAGES Bacteriophages or simply phages are the viruses that replicate within the bacteria Incase of certain phages, their DNA gets incorporated ita. the bacterial chromosome and remains there permanently. Phage vectors can accept short fragments of foreign DNA into their genomes. The indvoge ll phages fs that they eam take wp larger DNA segments than plasmids. Hence phase vettors are prefered for working with genomes of human cells Bacteriophage X Bacteriophage lambda for simply phage Al, 2 vius of Ecol has been most thoroughly studied and developed as a vector. In order to understand how bacteriophage functions as a vector, it is desirable to know its structure and life eyele (Fig. 69). Phage 2 consists of a head and a tilBIOTECHNOLOGY (both being proteins) and is shape is comparable to a miniature hypodermic syringe. The DNA, located in the head, isa linear molecule of about 50 kb. At teach end of the DNA, there are single-stranded ‘extensions of 12 base length each, which have cohesive (cos) ends. ‘On attachment with al 19 E.coli, phage 2. injects jas DNA into the cel Inside Ecol, the phage linear DNA cyclizes and gets ligated through cos ends to form a cicular DNA. The phage DNA has two fates tic cyele and lysogenic cycle. Lytie cycle + The circular DNA replicates and it also directs the synthesis of many proteins necessary for the head, tall etc, of the phage. The circular DNA is then cleaved (to form cos ends) and packed into the head of the phage. About 100 phage particles are produced within 20, minutes after the entry of phage into Ecol. The host cells then subjected to lysis and the phages are released. Fach progeny phage particle can infect a bacterial cell, and produce several hundreds of phages. It is estimated that. by repeating the lytic cycle four times, a single phage can cause the death of more than one billion bactenat cells. 17a foreign DNA Is spliced into phage DNA, without causing harm to phage genes, the phage will reproduce (replicate the foreign DNA) when it infects bacterial cell. This hhas been exploited in phage vector employed cloning techniques. Lysogenic eyce + In this case the phage DNA instead of independently replicating) becomes integrated Into the Ecoli chromosome and replicates alang with the host genome. No phage particles are synthesized inthis pathway. Use of phage i as a vector Only about 50% of phage 2 DNA is necessary for its multiplication and other functions. Thus, as much ae 30% (ie.p to 25Kb) of the phage DNA ‘an be replaced by a donde DNA for use in cloning experiments. However, several restriction sites are present on phage 2 which isnot by ise a suitable Yector. The I-based phage vectors are mecifiations ‘ofthe natural phage with much reduced number of restriction sites. Some of them are discussed hereunder Insertion vectors : They have just one unique cleavage sito, which can be cleaved and a foreign DNA ligated in, It is essential that suficient DNA a5 fe iaaat en fayiacerso Space rte forapn DNA at ak, Replacement vectors : These vectors have a pair of restiction sites to eave the non-essential ONA {stuffer DNA) that will be replaced by a foreign DNA. Replacement. vectors can. accommodate ‘vp 1 24kb, and propagate them. Many phage vector derivations insertion’ replacement) have been produced by researchers far use in recombinant DNA technology. The main advantage of using phage vectors is ‘that the foreign DNA can he packed into the phage lin vito. packaging), the latter in turn can be injected ito the host cell very effectively (Note : NNo transformation is required) Phage M,, vectors Phage Mya (bacteriophage M,,) is a single- stranded (of Ecoli Inside the hostel, My; synthesizes the complementary stand to form 1 double-stranded DNA leplicative form DNA; RF DNA\, For use as a vector, RF DNA is isolated and {foreign DNA can be inserted on it. This is then felurned to the host cell a8 plasmid. single- Stranded DNAS ate recovered fom the phage paticles, Phage Mis useful for sequencing DNA through Sanger’s method (Refer Chapter 7). cosmups CCosmids are the vectors possessing the characteristics of both plasmid and bacteriophage 2. CCosmids can be consiructed by adding a fragment of phage 2. DNA including eos site, t0 plasmids. A foreign DNA (about 40 Kb) can be insered into cosmid DNA. The recombinant DNA so formed can bbe packed as phages and injected into. Ecol (fig. 6.10. Once inside the host cell, cosmids bhehave just like plasmids and replicate. The advantage with cosmids is that they can carry larger Traginents of foreign DNA compared f0 plasmic. Phaside are the combination of plasmid) and phage and can function as either one (eas plasmid fr phage). Phasids possess functional origins. of replication of both plasmid and phage hand therefore can be propagated {as plasmid or phage) in appropriate Ecol, The vectors phasids may be Used in many ays in cloning experiments. [6 ___ a _|2 Ea aac neSE |pectaia te I Spe} eocend oO FSi Phage 2 T cin Ea Pari oom (oteosmi) Fig, 6.10 : Cosmics as vectors ARTIFICIAL CHROMOSOME VECTORS Human artificial chromosome (HAC) Developed in 1997 (by H. Willard human anificial chromosome is a synthetically produced ‘vector DNA, possessing the characteristics of human chromosome. HAC may be considered as a Selfreplicating microchromosome with 2 size fanging from Width to sth of a human chromosome. ‘The advantage with HAC is that it can carry human genes that are 100 long. Further, HAC can carly genes to be introduced into the cells in gene therapy. ‘Yeast artificial chromosomes (YACs) Introduced in 1987 (by M. Olson, yeast artical Cchromasome (YAC) fs 2 synthetic DNA that can BIOTECHNOLOGY accept large fragments of foreign DNA (particularly hhuman DNAI. iis thus possible to clone large DNA pisces by using YAC. YACS are the most sophisicated yeast vectors, and represent the largest capacity vectors available, They possess centromeric and telomeric regions, and therefor: the recombinant DNA can be maintained like 3 yeast chromosome Bacterial artificial chromosomes (BACs) The construction of BACS is based on one plasmid which is larger than the other plasmids tuned a6 cloning vectors. BACS can accept ONA inserts of around 300 kb. The advantage with bacterial artifical chromosome i that the instability problems of YACS can be avoided. In fact, a major part of the sequencing of human genome has been fccamplished by—using a library of BAC recombinant SHUTTLE VECTORS The plasmid vectors that are specifically designed to replicate in two different host say in E.coli and Streptomyces sp) ae refered to as shutle ‘vectors. The origins of replication fortwo hosts are ‘combined in one plasmid. Therefore, any foreign DNA tragment invoduced into the vector can be expressed in either host, Further, shutle vectors can be grown in ane hast and then shifted to another host ence the name shut). A good number of eukaryotic vecors are shutle veto CHOICE OF A VECTOR ‘Aman the several factors, the size ofthe foreign DNA is very imporant in the choice of vectors. The ficiency of this process in often crucial for etermining the suecess of cloning. The size of DNA insert that can be accepted by ditfecnt vectors is showin in Table 6.5. * Invvodocing a foreign DNA (i.e. the gene) into the cell isan important task in biotechnology. The cficiency of this process. is often crucial for determining the success of cloning. The most commonly employed gene transfer methods, rnamely transformation, conjugation, electo- poration and lipoection, and direct transfer of DNA ate briefly describedChapter 6 DNA size Phage zor ee Oven. Eco ay Pas atfcal Ecol 100-300 ehromasome (PAC) Bacal eicel Ecol 10-300 0 ‘hramsome (0) Yeast crovosome 8. eerie 0-700 1) TRANSFORMATION Transformation is the method of introducing forcign DNA ino bacterial calls (eg. Ecol). The ‘uptake of plasmid ONA by Ecol is caried out in ice-cold CaCl, (0-5°C), and a. subsequent heat shock (37-85"C for about 90sec). By this technique, the transformation frequency, which tetera tothe fraction of cell population that can be transfered, i reasonably good e3. approximately ‘one call for 1000 (10-) cel. Transformation efficiency : It refers to the umber of tansforsants per microgram of added DNA. For Ecol, translation by plasmid, the transformation efficiency is about 107 0 108 eels Ber microgram of tact plasmid DNA. The Bacterial ces that can take up DNA are considered § competent. The competence can be enhanced By altering growch conditions. “The mechanism ofthe transformation process not fully undersiod, t's believed thatthe CaCl, affects the cell wall, breaks at localized regions fad is also respansble for binding of UNA to cel Surace, A brie heat shock (ie. the sudden increase In temperature fom 5 to 40°C! stimulates DNA Uptake. In general, large-sized ONAS are less efficient in vansoxming. Other chemical methods for transformation Calcium phosphate (in place of CaCl) preferred for the transler of DNA into cultured = Gall. Sometimes, calcium phosphate may result in precipitate and toxicity to the cells. Some workers INTRODUCTION TO GENETIC ENGINEERING. we eran (D1 rf DNA transfer consucTION Conjugation isa atral_ microbial recombination process. Duting conjugation, RO live bacterts—a-donor and recipient) come tute, join by cytoplamie bridges ond tonfor single-stranded ONA’ om donor to recipient. Inside the recipient cell, the new DNA may integrate withthe chromosome (ather eae) 0° may remain foe (as isthe case with plasmids) Conjugation can occur among the cells fem different genera of bacteria (2 Salmonella and ‘Shigella cll). This i in contrast transformation “which takes place among the cells of a bacterial tenus, Thus by conjugation, transfer of genes from ‘0 different andl unrelated bacteria is possible, The natural phenomenon of conjugation is exploited for gene Wansfer This is achieved by transfering plasmid-inser ONA from one cell 19 another In genera, the plasmids ack conjugative finetinne and therefore they are not as such ‘capable of transferring DNA tothe recipient cell However, some plasmids with conjugative properties can be prepared and used ELECTROPORATION Electroporation is based on the principle that high voltage elec pulses can induce cell plasma membranes to fuse, Thus, electroporation is a technique involving electric _field-mediated ‘membrane permeabilization, Flecvic shocks can ‘iso induce eallular uptake of exogenous DNA Ubelieved to be via the pores formet! by electic pulses) from the suspending solution Flecttoporation is 2 simple and rapid technique for introducing genes into. the cells_itom_satious rsonme feroorganiens, plants and The besie technique of electroporation for transfering genes into mammalian cell is depicted In Fig. 6.11. The cell ae placed in a solution Coniaining DNA and subjected to electrical shocks fo cause holes in the membranes, The foreign DNA fragments enter though the holes into. the ‘ytoplasm and then to nucleus. Electroporation isan effective way t0 transform coli cells containing plasmids with inser DNAS longer than 100 4b, The transformation efficiency is aroun 10° ansformants per microgram of DNA° ° Bangs ie oo é oo oF im cae DONA taprens per So- é coe 2S oo 8 ial ae. al seat eee as co oO Po yates et rough oes ON Fig. tt: Gone raster by tetroporation. (ote : Magnan depts ond. for small plasmids (about 3ks) and about 10° for large plasmids (about 130 Kb) LIPOSOME-MEDIATED GENE TRANSFER Liposomes are circular lipid molecules, which have’ an aqueous interior that can carry nucleic acids. Several techniques have been developed to fencapsulate DNA in liposomes. The liposome: mediated pene transfer, referred to as lipoection, is depicted in Fig. 6.12 ‘On treatment of DNA fagment with liposomes, the DNA pieces get encapsulated inside liposomes These lipasomes ean adher to cell membranes and BIOTECHNOLOGY fuse with them to transfer DNA fragments. Thus, the DNA enters the cell and then to the nucleus. The postvely charged liposomes very eficiently complex With DNA, bind ta cells and transfer DNA rapidly. Lipofection isa very efficient technique and is used for the transfer of genes to bacterial, animal and plant cals Thelen meio of yerte waste ate belly described above. More details are given in the respective chapters applying these techniques to achieve gene transfer TRANSDUCTION Sometimes, the foreign DNA can be packed inside animal viruses, These vieuses can naturally posse LUposame with ONA, A oot Nucleus(Chapter 6 : INTRODUCTION TO GENETIC ENGINEERING Infect the cells and introduce the DNA iota host cells. The transier of DNA by this approach is refered t0 as vansduction DIRECT TRANSFER OF DNA tis possible to dretty transfer the DNA into the all _nucone Micrsinjortion and. panicle bombardment are the twa techniques commonly sed fr this purpose Microinjection DNA transfer by microinjection generally used (or he cise cel Thi ecnique i aso Useful 1 moder DNA io ge elaichas cace, ees and thecal of ealyemby Some more deals on gene tansfer methods Including penile hombardment are given, Chapter 48. The tm traps i used forthe transer Diva into eubaryoue cell by varius physical oF chemical mest A clone refers to a group of organisms, cals, ‘molecules or other objects, arising trom a single Individual. Clone and "colony are almost synonymous Gene cloning strategies in. relation to recombinant DNA technology broadly involve the following aspects (Fig. 6.13, + Generation of desired DNA fragments + Insertion of these fragments ilo a cloning vector + Introduction of the vectors into host cel # Selection or screening of the recipient cells tor the recombinant DNA molecules. Irs obvious that fora good undersanding (or account of gene cloning strategies, tis chapter has ta be learnt in detail In alton, the reader must, Invarably cefer gene libraries (Chapter 9) for Cloning of genes and screening strategies, and the polymerase chain reaction Chapter 8) fo in vitro eneration a lame quantities of DNA. Further gone Cloning alto involves the expression of genes which is described uncer manipulation of gene expression in host cells (Chapter 1), (GENERATION OF DNA FRAGMENTS (Pestcion endonucesse gestion, cDNA yptheals, POR, cromical sy) INSERTION WTO ACLONING VECTOR talon eS cas sesve en, | hemepoimer ang ior maecis) INTRODUCTION INTO HOST CELLS | [Traneiomion, translation, vanedicion) ‘SELECTION OF SCREENING ‘Gibrazston, PCR, mmonochemcal ‘maths, protsn-protinracons, {netonal complementation) CLONING FROM GENOMIC DNA (Of MRNA? DNA represen the complete genetic material cf an organism which is refered to as genome. ‘Tagorticaly speaking, cloning rom genomic ONA is supposed to be ideal, Bul the DNA canains non coding sequences {inors), contel regions. and repetitive sequences. This complicates the cloning strategies, hence DNA a5 2 source materia isnot peered, by many workers. However, if the Sbjective of cloning is to elucidate the conttl of iene expression, then genomic DNA has t0 be “variably used in cloning ‘The use of mRNA in cloning is prefered fr the fotlowing reasons. ‘MRNA represents the actual senetic Information being expressed, + Selection and isolation mRNA is cas + As inttons ae removed during processing, mRNA reflects the coding sequence of the gene, +The synthesis of recombinant protein i easy with ‘mRNA cloning Besides the dliect use of genomic DNA oF MRNA, itis possible 1 symhesize DNA in the laboratory (Chapter 7), and use it in-closingrr Ai experiments. This approach Is useful ifthe gene Sequence is shart and the complete sequence of Amina acid is known, ‘The different strategies for the cloning. of genomic DNA and mRNA are described under tne libraries (Chapter ®) For additional information on the genetic ‘engineering of plants, the eader must refer Chapter 429. Sore details on the following aspects are given in that chapter + Gene ‘vansfer methods (vectormediated. gene transfer-—T DNA, plant viruses; dtect ONA transfer —physical and chemical methods + Marker genes for plant transformation. + Promoters and terminators. + Transgene stability, expression and gene silencing. + Chloroplast vansformation. With the success of Boyer-Cohen experiments (in 1973), it was realised that recombinant DNA technology could be used to create organisms wth novel genes, This created worldwide commotion {among scientists, public and government officials) about the safety, ethics and unforeseen Consequences of genetic manipulations. Some of the phrases quoted in media in those days are ahve. + Manipulation of lite + Praying God. + Man made evolution + The most theatening scientific research, ‘was feared that some new organisms, created inadvertently or deliberately for warlare, would Cause epidemics and environmental catastrophes. ‘Bue tothe fears of the dangerous consequences, 2 ‘cautious approach on recombinant DNA ‘experiments was susseste, BIOTECHNOLOGY In 1974, a group of ten scientists Jed by Paul Berg wote a letter that simultaneously appeared in the prestigeous journals-Nature, Science “and Proceedings of the National Academy of Sciences “The dangers of DNA technology were printed out in that leter (highligh given below) Recent advances in techniques for isolation and joining of segments of DNA now permit Construction of biologically active. recombinant DNA. molecules in vitro. Although such experiments ae likely to facilitate the solution of important theoretical and practical. biological problems, they wauld also result in creation of hnovel types of DNA elements whose biological properties cannot be completely predicted. There is Dvserious concem that some of these DNA ‘molecules could prove biologically hazardous. ‘The letter ako appealed to molecular biologists worldwide for a moratorium on many kinds of recombinant DNA research, particularly those involving pathogenic organisms. ASILOMAR RECOMMENDATIONS In February 1975, a group of 139 scientists from 17 countries held a conference at Asilomar, & Conference center in California, USA. They assured the uneasy public that the microorganisms used in DNA experiments were specifically bred and could ot survive outside the laboratory. These scientists formulated guidelines and recommendations for ‘conducting experiments in genetic engineering. NIH GUIDELINES: National Institute of Health (NIE), USA constituted the Recombinant DNA Advisory Committee (RAO) which issued a set of stringent ‘uidelines to conduct research on DNA. RAC was in fact overseeing the research projects involving gene oplicing nd recombinant DNA. Some of the imporant original NIM recommendations on recombinant DNA research relate tothe fallowing aspects. + Physical laboratory) containment levels for ‘conducting experiments + Biological containmentsthe host. into which foreign DNA is inserted should not proliferate outside the laboratory of wansfer its DNA into cother organisms,‘+ For research on pathogenic organisms elaborate, controlled and self-contained rooms were recommended ‘+ For research on less dangerous onanism, units eauipped with high quality filter systems should be used + No deliberate release af any organism containing recombinant DNA into the environment. It may ke noted here that although the NIH guidelines did not have the legal status, most insitutions, companies and scientists voluntarily complied Relaxation of NIH guidel Teas in 1980, the orignal NIM guidelines were considerably relaxed by NIH-RAC, based on the experience and experimental data obtained fom the NiM-sponsored studies on recombinant DNA research, It was almost agreed that the original apprehensions on recombinant DNA research were uniounded. fat that Ue yer engineering seals flourished and progressed rapidly after elaxation of NIH guidelines tmay however be noted that NIH- RAC continues to be a watchdog over the DNA technology experiment. Pharmaceutical products of recombinant DNA. As the recombinant ONA technology progressed, many pharmaceutical compounds of hhuman health care are being. produced trough genetic manipulations, Most counties consider that the existing regulavons for approval of pharmaceuticals of commercial use are adequate to ensure safety since the process by which the product manufactured. Is. irelevant. Thus, the Fecombinant DNA, product (protein, vaccine, drut] Is evaluated for its safety and eficacy lke any other pharmaceutical product, Genetically engineored organisms (GEOs) Recombinant DNA research has resulted nthe stains bearing endABIOTECHNOLOGY Calose bene wath got) ‘tations improve the stability and yield of plasmid DNA, PURIFICATION OF mRNA ‘Among the RNAS, mRNA is frequently required Ina pure fm for genetic experiments Alter the cells ate distuped on Isis by liferent techniques (See p. 99), the cellular extract is ‘deproeinised by treatment with phenol or phenol/ chloroform mixtures. On centrifugation, the nucleic acide get concenitated inthe upper aqueous phase which may then be precipitated by using isopropanol or ethanol ‘The purification of mRNA can be achieved by affinty Chromatography using oligo (T)-celluose (fig. 7. Ths is based on the principle that oligo {dT-cellulose can specially bind (0 the poly (A) tails of eukaryotic mRNA. Thus, by this approach, its possible to isolate mRNA from DNA, rRNA and INA. ‘As the nulleic acid solution is passed through an affinity chromotographic column, the oligoidT) binds to pola) tails of mRNA. By washing the column with high-salt buffer, DNA, #RNA and tRNA can be elute, while the mRNA is tightly bound, This mRNA can be then eluted by washing with low-salt buffer, The mRNA is precipitated with ethanol and collected by centrugation (Fig. 7-4, Separation of large chromosomes of eukaryotes dabcbipoabll by canetenali detest ak The india chromosomes of evlaryots can be Separated by furescences ACS), ako now fl eytometry- oo Karyotyping. FLUORESCENCE-ACTIVATED CELL SORTING To cary out FACS, the dividing cells (ith condensed chromosomes) are carefully. broken ‘open, and a mixture of intact chromosomes is prepared. These chromosomes are then stained with fluorescent dye. The quantity ofthe dye thot binds te a chromosome depends on is size. Thus, larger ‘ehromosomes twit more DNA bind more dye and fluoresce more brightly than the smaller ones. The dye-mixed chromosomes are diluted and passed through a fine aperture that results in the formation of @ steam of droplets. Each droplet contains a single chromosome. The fluorescence of the chromosomes is detected by a laser. When the fluorescence indicates that the chromosome illuminated by the laser is the one desired,‘wh ye) ©2968 0000 Sat een OC fe ites ©0009 980 08 ical charge is specifically applied to these {and no others} waich get charged. This in the deflection of the dropless with the chromosome which can be separated from and collected (Fig. 7.5), Uncharged droplets m (Chapter 7 = BASIC TECHNIQUES IN GENETIC ENGENEERING 97 that do not contain the desited chromosome pass through 2 waste collection vase. COLLECTION OF CHROMOSOMES WITH IDENTICAL SIZE ‘The direct application of FACS (described above) 's not suitable for the separation of chromosomes sith identical sizes. eg, chromorormes 21 and! 22 in humans. Collection of such chromosomes can be achieved by use of special dyes eg. Hoechst 33258 and chromomycin Aj) which bind to INTrich DNA or GC-rich DNA. The rest of the Procedure isthe same that has been described It Is convenient to separate two or more chromosomes with identical sizes by diferent dyes This is possible since no two chromosomes are likely to coniain identical GC/AT contents Blating techniques. are_very_ widely used analytical tools forthe specific Keniieation of desired DNA or RNA fragments from thousands of molecules. Bloting refess to the process of immobilization of sample nucleic acids or solid support (nizocellulose or nylon membranes), The blotted nucleic acids are then used as targets in the hybridization experiments for their specific detection. An outline of the nucleic acid blotting technique Is depicted in Fig. 7.6. ‘Types of blotting techniques ‘The most comanly used blotting techniques are Usted below + Southern bloting (or ONA) None: Laing ho RIA) + Dot blotting (DNAVRNA) + Colony and plaque hybridization (cells from «colony : ‘The Souther blotting is named afer he scientist Fd Southern (1975) who developed it. The other ames Northern blating and Western blotting are laboratory. jargons which are now accepted. Western blotting involves the transfer of protein blots and their identification by using specific antibodies.BIOTECHNOLOGY Immobilization of nucle acids ‘Soe Bot (ONA ‘Noror Ble (RNA) Dots (ONAIANA) CCoeny and plaque bt (Cals em coiony) Prahyseaton| Labeled DNA or RNA probes yer Siegen wastes Fie, 78 + An autie of th 1A diagrammatic reprivntation of = typical blotting apparatus is depicted in Fig. 7.7. SOUTHERN BLOTTING Southern blotting technique is the first nucleic ‘acid bloting procedure developed in 1975. by Souther, It is depicted in Fig. 7-8 and briely described | cian las plate 4 Paper sues “The genomic, DNA fsolted from calsissues i gested with one o¢ more restriction enzymes ‘his mistureis loaded ito a wel in an agarose oF polyacrylamide gel and then subjected to ee ‘Genomic DNA rele ae 1S wprzed bands ma = eeChapter electrophoresis. DNA, being nezatively charged migrates towards the anode (positively charged electrode); the smaller DNA fagments move faster The separated DNA molecules ae denatured by exposure 10a mild alkali and vansfered to nitrocellulose or nylon paper. This results in an exact replica of the pattem of DNA fragments an the gel. The DNA can be annealed to the paper on ‘exposure to heat (20°C). The nitrocellulose or nylon paper Is then exposed to labeled cDNA probes, These probes hybridize with complementary DNA molecules on the paper ‘The paper after thorough washing is exposed to X-ray film to develop autoradiograph. This reveals specific hands coresponding tothe DNA fragments recognized by cDNA probe, Factors affecting Southern blotting 1. Stringent conditions (stringency control) : ingency refers to the specificity with which a cular DNA target sequence is detected by a je. Ths, with high. stringent conditions levated temperature and low salt concentration) ly completely complementary DNA sequences il bind and hybridize. However, low stringent tions will allow hybridization of partially fed sequences. Therefore, strnwency contol very essential for specific detection of DNA ecules in Southern blotting. With good control stringency, itis now possible to detect a DNA cule just witha single base pair diference 2. Membranes for blot transfer Inthe early 5 nirocellulose was Used for immobilization of molecules diring blot transfer. The crawoack ritacellsose is thet iis fragile and has to be ly hapa In recent years, ost laboratories non membranes as they posses high tensile nd_beter binding capnity.for_nuceic ons of Southern blotting em bloting technique is exeraly specific nsive, although It a simple technique the applications are ited an invaluable method in gene analysis 3 for the confimation of DNA ening for mapping restriction sites around a ceopy gene sequence. UES IN. GENETIC ENGENEERING SRE RNA sxract a + rorenscally applied to detect minute quantities ‘of DNA (to identify parenthood, thieves, rapists ete + Highly useful forthe determination of resrction fragment length polymorphism (RFLP) associated ‘ith pathological conditions. + DNA pieces from one species (es. human) can bbe used to detect DNA molecules from related species (eg, chimpanze, cow). Ths technique is feferted to 95 Zoo-blotting. NORTHERN BLOTTING. Northern blotting isthe technique for the specific identification of RNA molecules, The procedure adooted is almost similar to that ‘escrbed for Southern blotting and is depicted in Fig. 7.9. RNA molecules are subjected to electrophoresis, followed by blot transer, bybridizaton and autoradiography. RNA molecules donot exsily_ bind 10 nitroclose paper or nylon membrane, Blot transfer of RNA maces carted ot by wing 2 Chemicals reactive paper prepared by ‘iazotiaion aba ase ‘lazabeneylonmetyl(O8N0 paper. The RNA can Covalent bind to"DBM papet4100 Some warkers have later developed appropriate conditions t0 blot RNA bands on nitocellulose paper, and mecified nylon membranes, These-are Fotw widely used in RNA blating. The use of DBM papers is almost discontinued. The blottransered RNA molecules hybridize with DNA probes which can be detected. by Autoradiouraphy. ‘Northern blotting is theoretically, 2 good technique for determining the number oF genes {Ghrough mRNA} present on a ven DNA. But ths is not really practicable since each gene may give Fise to two or mare RNA Wanseripis. Another drawback isthe presence of exons and introns. DoT-BLoTTING Dotblotng is 2 modification of Southern and Northern blotting techniques described above. In this approach, the nucleic acids (DNA or RNA) are Pees ho ae {-lectophoress. The Fybrdization procedure Is the same 35 in orginal blotting techniques Dot-blating technique is particularly useful in fobiaining quantiatve ata for the evaluation of gene expression. WESTERN BLOTTING ier barrie eae he 3% to very neil 4 undead oe Reclec eid functions, particulary during the cae Seco F€ technique of Western bioting involves the traner of lecnoghoresed” tein bands from ply Fylon oF “itocellulose ‘These proteins can be deected by sec proteins actions, adibadies ot lectins aré commonly use for this purpose. a Autoradiography is the process of localization and recording of a radiolabel within 2 solid Specimen, with the production of an image in 2 photographic emulsion. These emulsions are composed of silver halide crystals suspended in selatin. ‘When a Pepatile or « yay fom a radiolabel passes through the emulsions, siber ions are BIOTECHNOLOGY converted to metallic silver atoms. This resus in the development of a visible image which can be easily detected, Direct autoradiography Direct autoradiography is ideally suted for the tection of weak t0 medium strength Bremiting ‘adionuclidas PH, MC, 35), In this technique the sample is placed in dect contact with the film. ‘The radioactive emissions rest inthe development of black areas Indirect autoradiography For the detection of hishly energetic P-bartices (ea 28 1251, leet autoradionraphy is not suitable, This Is because these emissions pass through and beyond the film, and a major part of the eneray ges wasted Indirect autoradiography is. useful for the detection of highly energetic Brpatcles. In this technique, the B-particle energy’ fist converted to light by a scinlatr, which then emits photons on ‘exponure to photographic emulsion. Applications of autoradiography As already described, autoradiography is closely associated with blotting techniques for. the detection of DNA, RNA and proteins. COLONY AND PLAQUE BLOTTING Colony and plague bloting is a_process of hybridization for the specific identification and ‘purification of colonies, bacterial clones). This Technique is depicted in Fig. 7.10, and briefly described hereunder. The desired bacteria are grown as colonies on an agar plate. When a nitrocellulose fier paper is tveriid on the agar plate, the colonies get transfered, Thy are permanently fixed tothe paper bby heat. On teatment with alkali (NaOH), the cells lyse and the ONA gets denatured. When these DNA prints are exposed to -speciic probes (radiolabel), hybridization occurs. The hybrid complex canbe localized and detected by autoradiography. ‘A similar strategy (described above for bacteria) can be used forthe identification of phages and fragments of phage libraries(Chapter 7 : HASIC TECHNIQUES IN GENETIC ENGENEERING 10 :Golony and plaque biting tenia. termination of aucleotide sequence in a DNA ule fs the basic and fundamental requirement biotechnology. DNA sequencing is important to stand the functions of genes, and basis of ited disorders Further, DNA cloning and gene lation invariably require knowledge of ate nucleotide sequence MAND GILBERT TECHNIQUE ‘The fst DNA sequencing techniave,_using feeds ese meter Paramore 404 ‘Dabieatanded DNA i ‘Sng-avandoa| ONA Tabeo0) ana ue Dee Ra Pe aesle = eae - ye cd eames Ge El eneeer: To AVORES vs ent (Spat ses ented ns regret mes Fer eens feos) eee fee = 5 |e - f - - je =e - - 3 = 1 Sie | - A i - 7 snot = a ar amas ATACTGOGACT Sequoncod rand TATGACGCTGA Complementary stand Fig. 7. ‘Maxam and Gilbert method fr DNA Gilber (1977). This method is briefly described Below (Fig, 7.10, A strand of source DNA is labeled at one end with 220. The two strands of DNA are then sepailted. The labeled ONA is distbuted ino four102 samples in separate tubes). Each sample is subjected. to. treatment with a chemical. that specifically destroys one (G, C) or two bases (A+, T +0) in the DNA, Thus, the DNA strands are partially digested in four samples at sites G, A+G,1+C and C. This results inthe formation ‘of a series of labeled fragments of varying ents. ‘The actual length of the fragment depénds on the site at which the base s destroyed fom the labeled ‘end. Thus for instance, if there are C residues at positions 4, 7, nd 10 away from the labeled end, then the treatment of DNA that specifically destroys C will give lebeled pieces of length 3, 6 and 9 bases. The labeled ONA fragments obtained in the four tubes are subjected to electrophoresis side by sie and they are detected by autoradiograph, The sequence of the bases in the DNA can be constructed rom the bands on the electrophoresis DIDEOXYNUCLEOTIDE METHOD Currently, the prefered technique for desermining cleotide sequence in DNA is the fone developed by Sanger (1980). This is an enzymatic procedure cormmonly refered to a8 ideonynucletice method or chain termination ‘method (Note : Fredrick Sanger won Nobel prize ‘wige; once fr determining the stucture of protein, insulin; the second time for sequencing. the nucleotides in an RNA virus A dideoxynucleatide is a. laboraton-made chemical molecule that lacks a hydroxy group at both the 2” and 3” carbons ofthe sugar (Fig. 7.12, This is in contrast to the natural deoxyibo- ‘nucleotide that possesses at 3° hycroxy group an the sugar ‘Termination role of dideoxynucieotide Inthe normal process of ONA replication, an Incoming nucleoside triphosphate is attached by its [phosphate group to the 3-hydroxy group ofthe last nucleotide ofthe growing chain (Refer Chapter 3) when a dideoxynucleatide s incorporated to the rowing chain, no further replication occurs. Tiss Because — dideoxynucleotie, lacking a 34hydroxyl group, cannet form a phosphodiester bbond and thas the DNA synthesis terminates, Sequencing method The process of sequencing DNA by dideoxynucleotide method is beelly described. A BIOTECHNOLOGY eoeod Base 8 cS Woo.7 Aw fA e000 & Base "i Roan \a w/t single-stranded DNA to be sequenced is chosen as ' template tis attached toa prime (a short lenth ‘of DNA oligonucleotide) complementary to a small section ofthe template. The 3”-hydroxyl group of the primer initiates the new DNA synthesis NA synthesis i cated out in four reaction tubes. Each tbe contains the primed DNA Klenow Subunit he larger agment of ONA polymerase of E colt, four dideoxyibonuclatdes (dATP, ICIP, AGT oF dATTP. Ie 8 necessary to tadolabel (wth 222 the primer or one of the eoxyribonaceaties As the new ONA syhess completed, each ‘one of the tubes conains fragments of DNA of ‘arying lenth our to primer. Let us consider the first reaction tube with dideonyadenosine (dATP. inthis be, ONA syrhess terminates whenever the growing chain incorporated ddA {complememary wo oT on the template sara. ‘Therelore, his tube wll contain a series of diferent Teng DNA fragment, each ending with A. Tn sma fasion, or the other 3 teachin tubes, DNA synthesis tos ashe respective dieoxynucleties ae inconporate The synthesis of new DNA Hagen inthe four tubes i depicted in Fig. 7.1. The DNA pieces are denatured to yield ree stands with radiolabel The samples from each {ube are separated by polyacrylamide gel electrophoresis This separation technique resolves DNA pies, diferent in size even by 2 singleChapter 7 = BASIC TECHNIQUES IN GENETIC ENGENEERING 103 Template s acaTecaars eee . pamar OM Newly synthesized DNA wth dconymnootd Prime nego” Preeti nant ate Pree Primer-ooTAeA. Pre © 8 Primar-OGTABCTTEdA acre Primer 1 Primer 6 Pamer #8 Primer OOTAGBEC escrP Primer 2 Primer Cada Primers 5 Prmer-O6TAdIG “aarre Primers 9 Prec Primar+7 Peer OSTAGCHBT Primers 8 Primer OoTAGCTERE nucleotide. The shortest ONA will be the fastest moving on the electrophoresis. The sequence of bases in 2 DNA fragment is determined by identifying the electrophoretic (radiolabeled) bands by autoradiography. In the Fig. 7.14, the sequence of the newly synthesized [DNA fragment tat i complementary to the orignal DNA piece is shown, Its conventional to read the bands rom bottom to top in 5’ 3" direction. By ‘noting the order of the bands first C, second G, third T and s0 on, the sequence of the ONA can be determined accurately. AS many as 350. base Sequences al @ DNA fragment can be clearly identified by using autoradiographs, “Modifications of dideoxynucleotide hod Replacement of 22P-adiolabel by 33P_or 295. oe ere INA polymerase of the thermoptilic bacterium, acu in place of Klenow fragment of [col DNA polymerase |) or a modified form of T7_DNA polymerase (sequenase) Improves ‘oon of aasonnucotioe be ake fe Limitations of dideoxynucleotide method There are mainly two limitations. in this techniques, The need for a singlesiranded DNA. template and the use of a primer to an unknown seajuences. These problems can be overcome by using bacteriophage ty BACTERIOPHAGE M,, AS A CLONING AND DNA SEQUENCING VECTOR The life cycle of phage Mys is depicted in Fig. 7.15. this virus contains 'a single-stranded circular ONA. When i infects €. col, the double stranded DNA is sythesized which isa replicative form (RF). RF DNA replicates until 50-100 copies are produced. Now the mode of replication is Changed 10 synthesize singlestranded | DNA [ssDNA] molecules. Each of the ssDNA of phage IMjs is coated with a protein. The phage particles then freely pass through the membrane to be Feleased out. Thus, the &, coli cells infected with IMyy can continuously secrete infective particles, Without undergoing lysis408 ans? wer Lagast + Smalet BIOTECHNOLOGY cP BETTE Sequence 2 a — 1 — t 8 “Fg 7.14 Soquonce ofa nowy syrneseed OWA buyin (eonplementry f engine! ana Bacteriophage My3 is employed as 2 cloning and DNA sequence determining, vector This 5 posable since the double-sanded replicative form can be isolated and used as a plasmid, while the Single-stranded phage DNA can be employed as 2 template for DNA sequencing. Usually, a target DNA with less than 500 bp can be cloned and sequenced in phage Ms ‘This dauble-sranded RF forms of phage My re Isolated and the ONA toe cloned i inserted. This ‘age which is comparable to a plasmid is returned fo € coll. (The uptake of phages with inserts can be screened by using Tac Z marker. As the phate Undergoes is lifecycle (Fig. 7-13), single-stranded [DNA molecules ofthe inserted DNA are recovered Sequence determination of the singlesvanded DNA can be done by dideexynucleotide method (See p. 102). The primer used is complementary to a part of phage M,, DNA which if close to the point of insertion, Therefore, a single primer can be {sed for diferent DNA molecules inserted, For determining the sequence of a larger DNA {= 2000 bp, it & necessary to clone diferent pieces of the DNA. From the sequences of these fragments requently overlapping), the final sequence can be built DNA SEQUENCING BY PRIMER WALKING Primer walking or primer extension technique is emplayed for sequence determination of long pieces of DNA (2 5,000 bp). In this method, plasmid cloned ONA containing target DNA is annealed with a prime (synthetic oligonucleotide) The primer binds with the DNA close to the Inserted DNA, Now the target DNA (250-350 nucleotides) is. sequenced, most fequenty. by tideosynucteotide method (described already) From the hase sequence of a fragment of the target DNA (determined in the first round), the second primer is chose. It's an oligonuceatie to bind to a region approximately 300 nucleotides ‘away from frst primer binding. The next fragment Of target_DNA can be sequenced by another 250-300 bases (second round). This sequence will be useful to choose the third primer and determine the next 250-350 bases hid round). In this fashion, the primer walking i continued unt the7: BASIC TECHNIQUES IN GENETIC ENGENEERING 405 0 Double. sanded BNA, 50-100 cai (Gouble- sands BNA) ‘ofthe marker, This inital probe will hybridize only imer walking technique for DNA sequencing with the clones containing fragment A. This depicted in Fig. 7.16 fragment A can be isolated, cloned and used aa probe to detect fragment 8. This procedure of OMOSOME WALKING IN DNA cloning and probing with fragments is repeated JENCING. again and again untl fragment D hybridizes with fragment € Chromosome walking is a DNA base sequencing ed in which the chromosome is analysed by As is evident from the above description, ing one Upto reach the othe. ne technique chromosome walking Uses probes derved fam the ly involes systematically moving along the ends of overlapping clones to faciliate a walk from a known location to an along with the DNA sequence. in the process of Tocation, and thus determining the DNA. ths walk, the sequence of the desired taaet gene The method adopted in chtomosome can be identified is depicted in Fig. 7.17, and. beelly 4 beiow, Chromosome jumping JA fragment with approximately 40,000 An improved strategy of chromosome walking is airs can be sequenced, To start witha marker chromosome jumping, Many regions of DNA that Toiidentiy the appropiate gene probe rom are dificult to be cloned by walking can be probe libra). This gene probe must have jumped. The procedure involves the circularization the base pai identical tothe base pais of large. genomic fragments generated by the106 BIOTECHNOLOGY ——————————EE Plasmid ‘Cones DNA Na ees eee ee ee Tr foot ® ERE © . Fig. 238 | Primer waiting tung in DNA sequencing (A) Mot round (B) Sooand round (6) This Pp PoPrmers 1.23 igestion of endonucleases. This is followed by (a) Cloning of the region lowering the closure of the Linke ke fragment. By this approach, the DNA sequences "etter 1 wor? located at far off places can be brought together ‘and cloned. chromosome jumping can be Cconstcted in this manner and used for long distance chromosome walks. Applications of chromosome walking ‘and jumping ‘The gene responsible for the disease cyte fibrin (CP) lua lee Hetifed by chromosome walking and jumping. The gene of protein responsible. for CF is called cystic fibrosis ttansmembrane conductance regulator (CFTR) and is located on the long arm of chromosome 7. AUTOMATED DNA SEQUENCING DNA sequencing inthe recent years is carted ‘out by an automated DNA sequencer. In this technique, flourescent tags are attached to chain- terminating nucleotides (dideoxynucleotdes). ThisChapter 7 tag_ gets incorporated Into the DNA molecules, while terminating new stand. synthesis. Four diferent fluorescent dyes are used to identify chain- terminating reactions in 2 sequencing gel. The DNA bands are separated by electrophoresis and etected by their fluorescence. Receniy, four dyes that exhibit strong absorption in laser ae in use fr fiutomated esquencins Advantages of automated sequencing = tis 3 rapid and accurate technique. Automated DNA Sequencer can accurately sequence up to 100,00 nucleotides per day. The cost works out to be not more than $0.2 per nucleotide. Automated ONA Sequencing nas ‘been successiully used in the human genome project. Some groups of research workers have levcloped sltemate mathods to Sanere method for ‘ejuencing of DNA, Untoaunately, despite the Initial excitement, most of these methods have disappeared from the scene. There are atleast 40 ethos with some promise for DNA sequencing _pyrosequencing, and gene chips microarrays) ROSEQUENCING Pyrosequencing is a DNA sequencing. method Is based on the principle of determining which ‘of the four bases (A. GC. Tic incorporated ich step while a DNA template Is copied. In Technique, the template copies ina straight aid -manner_whithout added dideoxy: ides (4ANTPS). As the new strand Is ized, the order in which the deox)- ides (GNIS) ate incorporated Is dewcied, Sequence can be fead_a5 the reaction (Fig. 7.18), The identification of base becomes possible since the addition of a is accompanied by the release of a ‘of pyrophosphate, This can be detected luminescence technique [procedure of pyrosequencing, each ONTP Fndividually (not all four together), slong dase enzyme. This enayme degrades Ff iti not incorporated nto the newly DNA strand. Once the appropriate BASIC TECHNIQUES IN GENETIC ENGENEERING. 107 TNA ete Prine exp MideotSS graded cere Meat vonacea act. pore TTT Tanta tic + pe Sulu Nucteotdase, rrp MSEPHEBES + Doped ee = eo | rucletide is Incorporated Into the new ONA strand, a molecule of pyrophosphate Is released ‘hie can be converted by the enzyme sulurylase into a flash of light (cheaniluminescence), By the addition af each ANTP separately one ater another, the order of the nucleotides added to the growing, [DNA strand canbe followed, andthe sequence can be identified, Detection ofthe molecule pyrophosphate isthe ‘prrescyucncing. Since pyrosequencing docs not Fequie electrophoresis of any ether DNA fragment Separation technique, its more rapid than chain termination sequencing The alo Unto of prseutnlng at is ible to dete! he squence of about 20 foci, This i mech tae an Oe Same Se eee eae Taree eda as var cane an motes tan beseqencd.Infac an aor ise ees occa aDNA CHIPS (MICROARRAYS) DNA chips or DNA microarays are recent developments for DNA sequencing as result of ‘ndvances male in automation and minarzation, A lanfe number afONA probes, each one with Siferent sequence, ate mobilized t-deined positions on the solid surface, made up of either. fiybar or los. Tee probes canbe abort ONA tmolecules sich as_¢DNAS~or-symthetic_oigo- ingles 2B Tor the preparation of high density arrays, ‘aliganucletides are syethesized_ in sity on the Surface of las oF slicon. This resuls in an ‘oligomucletide chp rather than a DNA chip. Technique of DNA sequencing ‘A DNA chip carrying an. aray of diferent “ligonuclaties can be used for DNA sequencing. For this purpose, a fluorescently labeled DNA test molecule, whose sequence is 10 be determined, is plied tothe chip. Hybridization occurs between the complementary sequences of the test ONA tnokzale and oigonuclesties of the chip. The postions of these hybridizing oligonucleotides fan be determined by confocal microscopy. Fach hybridizing oligonucleotide represents an ‘Benucleoide sequence that is present inthe DNA probe, The sequence of the test DNA molecule can be deduced rom the overlaps between. the sequences of the hybridizing oligonuclectides (Fg. 7.19) Applications of DNA chips “There have been many successes with this relatively new technology of DNA chips. Some of them are liste. ' Identification of genes responsible for the ‘ovelopmant of namau eystems. + Detection of genes responsible for inflammatory iseases, + Construction of microarrays for every gene in the ‘Genome of co and almost all the genes ofthe ‘yeast Saccharomyces corvsie BIOTECHNOLOGY ee aes) Fucreecnay tee inboa Su ypeszaton ri erase | aarooorr yer crocerta Tecra eboonveltes (6) ‘cocrrace) -“AGTOGOTTSSO-—- DNA sonuence Fig, 7.419: Wroaray (or eh) wetnoteay + Rapid detection of microorganisms for environmental monitoring. ‘The future of DNA chips ‘The major limitation of DNA chips at presents «fresno cer! sos n pokes hs “Ramat leon mar Rood hen identi + Detection and screening of single nucleotide polymarshisms (SNPS) that within the net few years such DNA chips wil be avalable. Ths will help the biotechnologiss to capture the functional snapshots ofthe genome in action for higher organisms.‘Advances in the laboratory techniques have made it possible to chemically synthesize ONA in a short period. Thus, oligonucleotides of about 100 buses can be produced in about 10. hours Laboratory symthesis of DNA Wecently By Use of DNA synthesizers or gene machines win specific Sequence of nucleotides, rapidly and inexensively, has significantly contibuted to cloning. Chemical synthesis of DNA is based on the ability to protect the reactive -5" and —3" ends by. blocking (protecting them, THE PHOSPHORAMIDITE METHOD. ‘The phosphoramidite method isthe technique of ce, curently in use, faethe synthesis of DNA, © synthesis carried out on a solid phase sytem by ataching the growing DNA strand toa solid ina reaction vessel, The procedure 1. Nucleoside attachment to solid support intial nucleoside base + sugar is alached at fend to an inert solid suppot, usually to alas with uniform pores, refered to as contlled flass (CPG) beads 2 Preparation of phosphoramidite: For each of the four bases (A, G, Cand weramidites canbe synthesized. This is by snaching dimethyl (OMT) group Stiydroxyl up of deoxyribose ard + piamine aoup tothe phos proup ucleosde- A ety ese roe Be jie woup. The geneal srucure of yamidite depicted in Fig. 720 pling : A nucleotce precuisor ies a amide) with is "phosphorus couples roxy ofthe intial nuclease bound to ion and deprotection : The unstable iphosphite is oxidized to pentavalent stable hiss followed by the removal of DMT deprotection) 5-hydeoxy! group. For oxidation and. deprotection are fogcther in Fig. 7.21 also, they are Independent reactions. (Chapier 7 : BASIC TECHNIQUES IN GENETIC ENGENEERING 109, "lg, 7.20 Siwotue of phospheramiata, (OurDimethonrty: Meet!) Another nucleotide precursor fs now added and the reactions described in steps 3 and 4 are repeated, oh Coupling, oxidation and_ deprotection are the the steps Th the cyclic reaction for the DNA synthesis by phosphoramidite method. The cycles sre rmpested again and again to ymthosi the desiced DNA. At the end, the completed oligonucleotide is removed from the glass support and the groups protecting the bases and phosphates are cleaved, Applications of synthesized oligonucleotides Chemically synthesized oligonucleotides have a number of uses in biotechnology 1.The linkers and adaptors are cligonucleotdes commonly employed in preparation of recombinant ONA (Chapter 6) 2. Chemically synthesized DNAS with known sequences can be used forthe syrthesis of proteins! polypeptides. 3. DNA probes, particulary the single-stranded oligonucleotide probes, ean be synthesized in the laboratory. This is based on the codon sequence of mRNA which in tum is dependent on the amino acid sequence. 4. Singlestranded oligonucleotides are used a5 primers in polymerase chain reaction (PCR). 5. For in vitro. mutagenesis, single-stranded cligonucleatides ae employee. the theae BIOTECHNOLOGY HOCH eset yee O crete abscess bak NUP Hg Prosshoramiate [pore Thana grneds of GNA y pigtail maid (0P6-Contoed por oat "ap ncnys Newey Neowin).Grass FSBASIC TECHNIQUES IN GENETIC ENGENEERING a = OH - OH P- 4 OW ‘OH P. 2. OH 6 ay 3 nesting 4 3 op 3 on T [eS eaais ese eee | | LoL | ie . 2 b ba + elo 8 ‘Ts DNA a towe on islet BI pee | | | a eee ‘Aasenne gone ‘ Fe 722 Canal anions fa aoe ‘ OF GENES Another way of synthesizing genes ist 4 double-stranded DNA; two single plementary oligonucleotides can be separately and on annealing they frm cs, This can be conveniently achieved ones (60-90 bp). However, the longer genes (> 300 bp) is associated Dractical dtculties. Ths is mainly due that the coupling efficiency for the Srthesis of DNA is never 100%, To this problem, small ONA Tragments are ‘and assembled (Fig. 7.22). Sealing of 1S achieved by use of T4 DNA ligase produce overlapping oligonucleotides which on annealing contain large gaps. These gaps can be filed by enzymatic synthesis of ONA by DNA, polymerase —while synthesizing. genes in the Taboratory, utmost cate should be taken to see that the nucleotides are in the correct sequence (this canbe checked by DNA sequence technique). ‘An Indian born and American seed scientist. Har Gobind Khorana and his associates were the fist to synthesize an entire RNA gene for yeast alanyl tRNA in 1972.he polymerase chain reaction (PCR) sa laboratory in vito) technique for generating large quantities of 2 specified DNA. Obviously, PCR is a celliree amplification technique for synthesizing multiple Identical copies (lions) of “any DNA of interes. Developed in 1984 by Kary Mullis faurate, 1993), Pretec 3 baie Oo acre Teese biologist. As isa photocopier a basic requirement Inan ofice, coi the PCR machine in a molecular biology laboratory! Principle of PCR, fe doublestranded DNA of intrest is denatured to separate into two individual sands. Fach strand is then allowed. to hybridize with a primer (renaturation).. The primer-template Suplex is used for DNA synthesis the enzyme DNA polymerase. These three steps— denaturation, renaturation and synthesis are repeated again and again to generate multiple forms of target DNA TECHNIQUE OF PCR The essential requirements for PCR are listed below 4, A target DNA (100-35,000 bp in length 2. Two primers (symhetic oligonucleotides of 17-30 nucleotides length) that are complementary to regions flanking the target DNA. 2. Four deoxyribonucleotides (dATP, dCTP, aT, rT, 4. A.DNA polymerase that can withstand at 2 temperature upto 95° C (he, thermostable The reaction mixture contains the target ONA, ‘wo primers (in exces), a thermostable DNA. polymerase (solated tom the bactenum termus Sauaticus (2, Taq DNA polymerase) and fout ‘deoxyribonucleoties. The actual technique of PCR involves repeated cycles for amplification of target DNA. Each cycle as throe stages. 1. Denaturation : On rlsing the temperature to about 95° C for about one minute, the DNA gets denatured and the to sPabdS separate 2. Renaturation or annealing + AS the temperature of the mixutre i slowly cooled to about 55°C, the primers base pair with the Complementaty regions flanking target DNA strands. This process is called tenaturation oF annealing. High concentration of primer ensures faanealing between each DNA strand and the frimer rather than the hwo seas of DNA 2, Synthesis: The initiation of DNA sythess occurs at 3-hydiowyl end of each primer. The primers ze extended by joing Te bases Complementary DNA sands. The synthetic process in FCR is gute comparable to the DNA Tepliation of the lacing seand Refer Chapter 3) However the temperature has 0 be kept optimal as requited by the enayme DNA polymerase For Tag 12(Ghapics 8 : POLYMERASE CHAIN REACTION [DNA AMPLIFICATION Denakraton| mn) und 32> Cl. The reaction can be stopped by lsing the iemperatue Go about 95°C) “The 3 stages of PCR in relation to temperature time are depicted in Fig. 7. Each cycle of takes about 3-5 minates, In the novral tice, the PCR fs cared ou in an automated hin, Asis evident trom the Fig. 8.2 cycle the new ne These new strands are refered to as long ‘and they sil be used in the second fhe second cycle uf PCR, Ue ONA strands + nowiy synthesized long template) are red annealed with rimers and subjected to ‘yess. At the end of second round, long and short templates (DNA stands with ‘sequence at one end, and. sequence nary to the othe end primer are formed. third cycle of PCR, the orginal ONA jong with long and short templates are the als, The techique of denaturation, ‘and. synthesis are repeated. This is repeated again and again for each 8 2 ¢ Sa : — eee [ SS ee ae oa <4 Long template Se ees a Lene template : L | "Fig, 82 : The pobmerase chain rancton (BCR) "epreeotng tne il tae ele (seni pres cycle. ts estimated that atthe end of 32nd cycle of PCR, about a millonfold target DNA. is synthesized (Table 8.0, The short templates ossessing precisely the target ONA as double- stranded molecules accumulate,Tara eng ominin ot ‘target DNA by polymerase chaln reaction Cycle number ‘Number of double- stranded target DNA 1 0 2 ° a i 4 4 5 8 6 6 7 2 6 6 128 10 8 " 52 2 08 13 oe 4 6 15 182 6 16964 7 2218 8 5508 19 sanor2 Ey a2 148 a s24288 2 1903578 2 2087 152 2 asota0e 5 ses 208 2% rezr7216 z sastace 2 er t08 886 a soazi.728 2 288495 56 st sieavosi2 ® 1.973741 24 BIOTECHNOLOGY, SOURCES OF DNA POLYMERASE In the original technique of PCR, Klenow fragment of coli DNA polymerase was used. This fenzyme, gets denatured at higher temperature, therelore, fresh enzyme had to be added for ‘each eyele, A breakthrough occurred (Lawyer 1989) With the itraduction of Taq DNA polymerase from thermophilic bacterium, Thermus aquaticus. The Tag DNA polymerase & heat resatant, hance tf thot necessary to freshly add this enzyme for each cycle of PCR. KEY FACTORS FOR OPTIMAL PCR Primers ; Primers play 2 significant role in determining PCR. The primers (17-30 nucleotides) without secondary structure and without Complementarity among themselves are ideal, ‘The complementary primers can hybridize to form primer dimer and get amplified in PCR. This prevents the multiplication of target DNA [DNA polymerase 2 As already described, Taq DNA polymerase is prefered as It can withstand tinh temperature In the hottaet protocol, DNA Te ere ded Sher te Feat denaton Jap of fe Hist cycle, This avoid te extension of the misiaTched primers that usually occur at low temperature Tag polymerase lacks proof reading exonuclease (95) activity which might contribute to erors in the products of PCR. Some other thermostable DNA polymerases with proot-eading activity have been identified ©, Tma DNA polymerase from ‘Thermotoga maritama; Pu DNA polymerase from reas nee Target DNA :In general the shorter the sequence of target DNA, the better isthe efciency of PCR, However, in frecent years, amplification OFDNA fragments upto 10 kb has been reported. The Sequence of target DNA i also important in. PCR. ‘Thos, Ge-sich regions of ONA stand hinder PCR. Promoters and inhibitors: Addition of proteins such as bovine serum albumin (BSA) enhances PCR by protecting the enzyme DNA polymerase. Humic eid, frequently found in archeological simples of target DNA inhibit PCR. ‘The basic technique of the PCR has been described. Being 2 versatile technique, PCR is(Chapter & : POLYMERASE CHAIN REACTION (DNA AMPLIFICATION) ous ‘Undesie ONA setts Por win, fest pers Sesand Gero 1 = PCR with, Sn ame “he secnd pie donot ans unger BRRence no BCH occurs Pa FI. 8 Nested peymerase chain action, z modified 2s per the specific demands of the situation. now cut with another resviction enzyme. This Thus, thete are many variations inthe orignal PCR, cleaves only the known sequence. The target ‘some of them are discussed, hereunder DNA so. formed contains the known sequence at both the ends"with target DNA at the middle NESTED PCR The PCR amplification can now be carried fut. It may be noted that the primers ae generated in the opposite direction to the normal, since the orginal sequence is inverted during cicolarization, Sequence similarities between the target DNA. and related DNA ate very frequently seen. AS a sult of this, the primers may bind to both the [DNAs and therefore even the undesired DNA als ANCHORED PCR amplifed_in_ PCR _Use of In the anchored PCR, a small sequence of nucleotides can be attached ragged) to the target Sineee i Wlesrated If DNA ie, the DNA is anchored. Ths is particularly ae ee eo Useful when the sequence surrounding the target both_from_target_DNA and undesired DNA. A, 5 i es OM ohana faa) I sletvely bind TO. tage DNA and Cp idO which see C_poME wed. The rng cn ao Be TORE DY Te Tt alps = As the adaptors possess a known sequence, the primey cai be chosen. /ERSE TRANSCRIPTION PCR ‘The PCR technique can also be employed for ig the amplification of RNA molecules in which @ resrichon endonuclease which does not eut case its veered to as everse transcription — PCR Known sequence but cuts the unknown (RT-PCR. For this purpose, the RNA molecule fe on ether side. The DNA fragments (mRNA) must be first converted to complementary sb are inverted and get circularized DNA (cONA) y the enzyme reverse transeriptase Tigase is employed as a. sealing agen). The EDNA then serves as the template for PCR. le containing: the known sequences is Different primers can be employed for the synthesis‘DNA wih known saqunce fn lou) Resvicten frndonucoase {estate | © © oO | cranes ) or of first sand of CDNA. These include the use of random primers, oligo dT primer and a. sequence specific primer (Fig. 8.5) ASYMMETRIC PCR CR echnique can also be tnd forthe shes of singlestanied DNA molecules, patcuaty tie SE-DRICTSencing Chater 7), te asymmetric PCR, io of 100 : 1 ire wsl,Ater 2007 oye DOR, Brig ‘haste, The result is that inthe nex5-10 PCR yeles, only single-stranded DNAS are generated REAL-TIME QUANTITATIVE PCR ‘The quantification of PCR products in liferent cycles is not as simple as projected by theretical considerations. (Table 8.1). In practice, large \atiations occur Ty4most commonly used technique for measuring the quantity of PCR is by employing a BIOTECHNOLOGY Ahgicence compound ie ethidium bromide. 6 pence stat Tr enablesanieONA mnecles bind to ebidium bromide which emit fuorescence that can be detected and DNA quantified, “The gyfithess of gemés by PCR and the role of ck in atedeced mutagenesis re described elsewhere (Reler Chapter 10) RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD) Normally, the objective of PCR is to generate Aefined fragments of ONA from highly specific primers nthe ronounced as api, Short coligonucleotide prime are arbitrarily sele “Fset_ of DNA fragments fandomly distributed throughout the genome. This technique, random amplified. polymorphic DNA is ako known as aril PCR (AP ‘The procedure of RAPD is comparable to the general technique of PCR. This method basically Involves the use of 2 siggle_pimer at_tow stringency. A single short oligonucleotide asually a 9-Tar Base primer) binds to many sites in the ‘genome and the DNA fragments are amplified frm them. The stingency of primer binding. canbe Increased after a few PCR cycles. This allows the amplification of best mismatches, RAPD can be Carefully designed so that it finally yields genome- specific hand patterns that are useful for Comparative analysis, This is possible. since ‘genomic DNA from two diferent individuals often produces diferent amplified patterns by RAPD. ‘Thus, a particular DNA fragment may be generated for one individual and nat forthe other, and this represenis DNA polymorphism which can he used ‘asa genetic marker. =) ama manaRAPD Is widely used by plant molecular biologists for the genetic identification of plant species Refs Chapter 53) Fors parpane, erent Combinations of nucleotides, most of them random Sligonucleotie primers have been designed and are Eommercally available, As each random primer Annals toa diferent region of DNA, many diferent fegions of loci on the DNA can be identified. RAPD Js thus useful forthe constuction of genetic maps id as a method for genomic fingerprinting tations of RAPD ‘The main problem of RAPD is associated with “epscduciili, I is often dificult obtain similar sof primer binding in dierent experiments. It therefore dificlt to correlate results obtained by ferent research groups on RAPD. IPLIFIED FRAGMENT LENGTH YMORPHISM (AFLP) AFLP_is_a very sensive method for detecting nciple oF rescton tagrien lerath poly pephicm (RFLP, Refer Chapter 14) and RAPD, LP may be appropriately egarded asa diagnostic ingerprinting technique’ that detects genomic resticton fragments, In the AFLP, PCR amplification rather than ther blating (mostly sed in REL) is used for on of resviction fragments. It may be ‘ated Tat AFLP i employed to detect the presence absence of restriction fragments, and not the ngths of these fragments. This is_the major rence between 1d RFLB. AFLP is very ‘sed in plant genetics. I hos not proved ful in the mapping of animal genomes, since is technique is mainly based on the presence of gh rates of substitutional variations which are not id in animals. On the ther hand, substitutional iio renltng in RFLPS are more common in Jars, “The basic principle of AFLP involves the plication of subsets of RFLPs using PCR Fe 56, __A genomic DNA is isolated and digested restriction with a6 bse pair ition site and Msel with a 4 base pair ition site. These two enzymes can cleave the (A and result in small fragments (¢ 1 Kb) which bbe amplified by PCR, Fortis purpose the DNA (Chapter 8 : POLYMERASE CHAIN REACTION (ONA AMPLIFICATION) ‘Eoameny sare rms | on rons oR adaptor | Meal adaptor arte: a Tae Aa amar wth one age sees. cma cant a [Adon of second fragments at ligated with EcoRI and Mset adaptors ‘These common adaptor sequences lanking genomic sequences) serve as primer binding sites ‘on the restriction fragments. The ONA fragments can be amplified with AFLP primers each having ‘only one selective nucleotide. These PCR products are dited and used as templates for the selective amplification employing two new AFLP primers that have 2 or 3 selective nucleotides, Aiter the selective amplifcation by PCR, the DNA products are separated on a gel. The resultant DNA fingerprint is identified by autoradiography.118 AFLP fragments represent unique positions in the genomes, and hence can be used as landmarks to bridge the gaps between genetic and physical maps fof genomes. In plans, AFLP is useful to generate high density maps, and to detect genomic clones RAPID AMPLIFICATION OF cDNA ENDS (RACE) ‘As akeady described (Seep. 115), reverse transcription, followed by PCR (RTEPCR} results in the amplification of RNA sequences in CDNA form, But the major limitation of RTEPCR is related to incomplete DNA sequences in cDNA. This problem is solved by using the technique rapid amplification of eDNA ends, RACE is depicted in Fig. 8.7, and briely described below. The target RNA is converted into a partial eDNA by extension of a ONA primer. This DNA primer 1vas first annealed at an interval position of RNA, hat toa far fom the Send of the molecule. Now addition dATP (As) and terminal deorynucleotiyl transterase extends the 3'end of the CDNA. This happens due tothe addition ofa series of As tothe FONA These Ae series nn act a the primer to anneal wo the anchor primer. second strand of DNA can be formed by extending the anchor primer. The daublestranded DNA is now ready for Smplifiation by PCR. ‘The above procedure described is called 5 RACE, since itis cartied out by amplification ofthe [Send of the starting RNA. Similar protocol can be used 0 cany aut 3°RACE when the ¥en RNA sequence is desied Limitations of RACE Since a specific primer is used, the Specificity of amplification of RACE may not be very high. Another disadvantage is that the reverse transcriptase may not fully reach the Sends of RNA, and this mis the uly of RACE. 1p recent yeas, some modifications have been done to Improve RACE. | eramon o | The advent of PCR had, and continues to have: ‘wemendous impact on molecular biology. The application of PCR are too many tobe listed her. Some of them are selectively and very briefly BIOTECHNOLOGY mRNA ar gaat. tein Wansterase 4” AAAAANNIN ‘Anne anche primer 15 NNNNANTTTTT 9° [AARAANNNN i ‘Second aang BAA Sythe Connie POR described. Other applications of PCR are discussed at appropriate places. PCR IN CLINICAL DIAGNOSIS The specificity and senativity of PCR is highly useful for the diagnosis of various diseases in humans. These include diagnosis of inherited disorders (genetic diseases), veal diseases, bacterial tiseases ec ‘The occurrence of genetic diseases frequently Ieniied by restriction “eagment. length poly- morphism (RFLP) can be employed only when there i a mutation esuting in a detectable change inthe length of restriction fragment. Many genetic diseases occur without the involvement of RFLP.Chapter 8 = POLYMERASE CHAIN REACTION (DNA AMPLIFICATION) 419 For all such disorders, PCR technique 1s a real boon, as it provides direct information of DNA. This is done by amplification of DNA of the relevant region, followed by the direct analysis of PCR product. Prenatal diagnosis of inherited diseases : PCR {employed in the prenatal diagnosis of inherited diseases by using chorionic villus samples or Cells from ammiocentesis. Thus, diseases like sickle-cell anemia, Brthalassemia and Dhenyiketonuria can be detected by PCR in these samples. Diagnosis of retroviral infections : PCR from {EDNA isa valuable tool for diagnosis and monitocing of retrovel infections, eg, HIV infection. Diagnosis of bacterial infections : PCR is used for the detection of bacterial infection eg, tuberculosis by Mycobacterium tuberculosis Diagnosis of cancers : Several virally-induced ‘cancers (eg, cervical cancer caused by human papilloma virus) can be detected by PCR: Further, Some cancers which occur due to chromosomal translocation (chromosome 14 and 18 in follicular Iymphoma) involving known genes are identified by PCR PCR in sex determination of embryos : Sex of human and live stock embryos feilized in vtto, an be determined by PCR, by using primers and DNA probes specific for sex chromosomes, Further, this technique is also useful to detect sex —linked disorders in fertilized embryos. PCR IN DNA SEQUENCING ‘As the PCR technique is much simpler and {quicker to amplify the DNA, itis convenient used or sequencing. For this purpose, singlestrands of DNA are required. In asymmettic PCR (See 1p 116), preferential ampliicaton ofa single-strand is caried out. In another method, strand removal ‘can be achieved by digesting one svand (usually done by exonuclease by its action on 5Ypho phoryated strand PCR IN GENE MANIPULATION AND EXPRESSION STUDIES ‘The advantage with PCR is thatthe primers need ot have complementary sequences forthe target DNA. Therefore, the sequence of nucleotides in a piece ofthe gene target DNA) can be manipulated and amplified by PCR. More details on this technique, refered to as site-directed mutagenesis, ate given elsewhere (Chapter 10). By using this method, coding sequence can be altered (hereby Changing amino acids) 10 synthesize protein of interest Further, gene manipulations are Important in understanding the effects of promoters, inators fc, in gene expression PCR is imporant in the study of mRNAs, the products of gene expression. This is carried out by reverse transcription — PCR, PCR IN COMPARATIVE STUDIES OF GENOMES The differences in the genomes of two organisms an be measured by PCR with random primers. The Products are separated by electrophoresis for ‘comparative identification. Two genomes. from closely related organisms are expected t0 yield more similar bands. For more details, reer the technique random amplified polymorphic ONA (ee p. 116), PCR is very important inthe study evolutionary biology, more specifically reiered ta as Dhylogenetics. As a technique which can amplify feven minute quantities of DNA from any source (hair, mummified tissues, bone, oF any fossilized ‘material, PCR has revolutionized the studies in palaentology and archaelogy. The movie ‘Jurassic Park, has created public awareness of the potential applications of PCR! PCR IN FORENSIC MEDICINE A single molecule of DNA from any source (blood strains, hair, semen et.) of an individual is adequate for amplification by PCR. Thus, PCR is very important for identification of criminals ‘The reader may refer DNA finger printing technique described elsewhere (Chapter 14). PCR IN COMPARISON WITH GENE CLONING PCR has several advantages over the traditional gene cloning techniques (Chapter 6). These include beter efficiency, minute quantities of starting material (DNA), costetectiveness, — minimal technical sil, time factor etc. In due course of time, PCR may fake over most ofthe applications of ene cloning! | he collection of DNA fragments (specifically genes) from a. paticular species represents ene libravies. The cretion or constuction of ene Nibraries (broadly genomic libraries) is accomplished by isolating the complete genome (enti DNA from 2 cell) which is. cut into fragments, and cloned in suitable vectors. Then the Speci clone carrying the desired arget) DNA can be identified, isolated and characterized. In this manne, a library of genes or clones (appropriately Considered as gene hank! for an the entre genome ff a species can be constructed, The sizes of Jgenomes in diferent species are variable (Table 9.4), A complete gene library for each organism contain all the genomic DNA Biotechnologiss are particularly interested in the isolation of genes (and therefore creation of ‘Organism Genome size™ (kb) Saectromyoascreiiae iasxtot “Tonsece text West s9xct0 ‘Drosophis melronater tact Mose ssi Hunan se ene libraries) which encode for proteins. There is 5 distin diference in the genes of prokaryotic and feukaryotic cells. In prokaryotic omganisms, the structural genes coding for proteins ar cootinuous. However, case of eukaryotes, the coding regions (exons) of structural genes are separated by non- coding. regions intons). For this reason, the “construction of gene ibrares for eukaryres Is more complicate, CREATING A GENE LIBRARY ‘The DNA from the source organism is digested by restriction endonuclease (eg, EcoR?, to result In fagmens. is desvable to crete conditions so that pani digestion and not complete digestion ‘cus, By this way, al posible DNA fragments of ‘arable size canbe produced. The paial digestion fof 4 ONA with a restriction endonuclease is ‘depiced in Fig 941. The cleavages occur at dliferent sites to result in DNA fragments of varying lengths, some of them may be lage while others are small. In practice, a combination of restiction heymes are ned to digest routes DNA to rleace 2 large number of DNA fragments. The desired Fragments can be isolated and cloned. Some workets use the term shotgun experiment (or shorgun approach) fo: cxaton of random clones; (without necessarily identifying all of ther) from a te(Chapter 9 = GENE LIBRARIES a RE = 421 re Fe Resticton endonuclease ‘feria qc Frere) 121 Paral gestion of @ DNA by th enzyme resistin endonuclease (RE) at ee ses {Note the caloured ragment represenis the dested gone and it ove (shown in) among several fragments formed. technique for ‘gene library technique developed by Maniatis al Jenses ane sed 10 target DNA. Partial digestion allows the Tajorty of DNA fragments with 2 10-30 Kb. The fagments are frequently and they can be Fractionated by gel esis he ssolated fragments ly 20 kb in size) arg Tagried fato 2 and cloned, ey ng @ gene library for humans an cellular DNA (the entire genome) subjected to digestion by restiction fs (e., EcoRI. The Tagiens formed are of about 4 kb size. (Le, 4000, fhases), Each human chromosome, ‘approximately 100,000 tb can be cut 5/000 DNA Fragments. As the humans have 23 clferent chromosomes (24 in man, there fare & total of $75,000 fragments of 4 Kb length formed. Among these 573,000 DNA fragments is the DNA or gene of interest (ay insulin gee) Now fs the selection of a vector and cloning process. Eco a harmless bacterium to humans is fost commonly used. The plasmids from E. coll are iuolated, They are digested by the same restiction 0 for cutting human fenome to. form “open plasmids. The human Chromosomal DNA fragments and open plasmids fre joined to produce recombined plasmids. These plasmids contain diferent DNA. fragments of humans, The recombined plasmids are inserted into coll andthe cells multiply (Fig. 9.2. The E. coll cells possess all the human DNA in fragments. I ‘must, however be remembered that each . coll ‘ell contains diferent DNA fragments. All the call cells put together collectively represent {genomic library (containing about 575,000 ONA fragments.BIOTECHNOLOGYChapter 9: GENE LIBRARIES ‘Other vectors for creating ‘genomic libraries Tn place of phages and plasmids, other “sectors are in use for constuction of lage sized libraries. These include cosmids, bacterial I chromosomes (BACS) and yeast artificial 18 (YACS), These ae considered high capacity vectors. Although they are for consruction of gene libraries, there Imany practical dificukies associated with their AS AN ALTERNATIVE TO :NOMIC LIBRARY CONSTRUCTION {As aleady described in deal (Chapter 8), PCR 4 technique for amplification ofa specific DNA nce. PCR with primers can be used to isolate et DNA directly from the genome. Thus, PCR a6 an allemative to ONA library (gene rary consruction by cloning. This s not always sible since PCR technique can be employed for plication of shor length ONAS (usually 1-2 Kb fio evaninnnn KL). ther, the igh rmperature used in PCR, sometimes causes Wamage to bases and generates nicks in DNA ‘rane. ‘Long PCR With some modifications in the PCR, it is ow possible to amplify ONA tragments up to a Tength of 22 Kb from the human genomic DNA, This & achieved by using a combination of two DNA polymerase enzymes, besides lowering the reaction temperature. One of the DNA polymerases has proofreading activity to remove the mismatched bases. Some commercial companies in fact provide enzyme cockals ideally suited for Tog PCR eg. TPs Lang PCR system marketed by ‘Stratagene. i contains Taq polymerase and the thermostable proofreading enzyme | Plse polymerase, Long PCR has been applied for the stuctured analysis of human genes and genames of HIV. Long PCR is unlikely to replace the construction ‘of genomic libraries. This is because creation of DNA libraries is permanent while long PCR is temporary. Further, PCR can be employed for ampliying soleced DNA fragments (of interes) fiom genomic libres, 423 Fragment ratios Biotechnologiss olen come across. minute quantities of staring materials eg, single cell, fixed sues, fossils etc. Is quite dificult to apply traditional technique for constuction of genomic libraries rom such samples. PCR is ideally sulted for isolation and amplification of genes from very small samples. Thus, PCR can be used for the creation of random genomic fagmant libeates. COMPLEMENTARY DNA LIBRARIES Cloning of eukaryotic genes is rather complicated and requires special techniques. This 's mainly due othe non-coding sequences (nrons) in the DNA. In the eukaryotic cell as the gene is transcribed, the RNA undergoes several changes in the nucleus (ferred to as splicing) to release a ‘mature and functional mRNA int the cytoplasm this manner, the introns ate removed, In some genes, introns form a major bulk ofthe gene. For Instance, in the human dystrophin gene, as much a2 99% of the DNA sequence Ts composed uf inrons. There are as many as 79 intros! ‘The prokaryotes, particularly the bacteria, do rot posses the bility to remove the introns. Hence the funetonal mRNA is not corretly formes in 3 prokaryotic cell for an eukaryotic gane. Thus, Cloning of eukaryotic genes becomes a ificult task, ‘Synthesis of complementary DNA Complementary ONA (eDNA} is a double stranded complement of an mRNA. CDNA can be symesized from mRNA by reverse transcrip ‘An eukaryotic functional mRNA which does not have introns possesses a G cap at 5” and a poly(A) tail at the 4° end lapproximately Zu adenine residues ‘The requisite mRNA is jolted and purified (panicularly fom cells which are rich in the Specific mRNA eg, pancreatic cells for insulin mRNA). An oligo-dT primer is added to bind tothe shor segment of polyA tal region (by annealing). This primer provides J-hydroxy! group Tor the synthesis of a DNA stand, With the addition of the enzyme reverse wanscriptase and four feoxynucleotides (AATF, dTTP, dGTP and CTR), DNA syathess proceeds, For the bases A, G, CandBIOTECHNOLOGY 56 ARAAAAS ANA [omen primer a ge SARAAAAGN yrtts Reverso tanscripase 5 sraaiTes OH AAAAAA, AAAAAA TTT a \-OH 3 | Lon 3 DNA pomaraso i oracoymerase ia [ten SaaNTeS AAAAAA ARAAAA ae TTT Nase H Nase ‘Sinucase Slnuslease ~ Complete cONA ‘complete ea Fig, 93 = Syniheas of complementary DNA (cDNA). U in the template (mRNA), the corresponding ‘complementary bases in DNA respectively are 1, GG and A. The newly synthesized first DNA’ stand has a tendency to fold back on to wel fr a few auclectides to form a haipin loop (Fig. 93. ‘The Joop of the fist DNA sand serves as the template for the synthesis of second DNA strand. By the addition of E.coli DNA polymerase (Klenow fragment, the second ONA strand synthesis occurs starting fom the end of the halpin loon On treatment with the enzyme RNase H, mRNA molecules are degraded, The enzyme SI nuclease ‘leaves the hairpin loops and degrades single Stranded ONA extensions. The final products are Complementary DNA copies of orginal mRNA, Some of them are complete while others are incomplete. Limitation of the technique : The main lsadvantage withthe haipin method isthe loss of cleavage by 51 please. Improved method for eDNA synthesis Jo overcome the limitation described above, some improvements have been made in cDNA synthesis, One such improved technique is shown in Fig. 94 As the fist strand of cDNA i synthesized, i is talled with cytidine residues with the help of the cefzyme terminal transferase. The mRNA strand is hydrolysed with alkali, and the ful length cDNA is recovered, A synthetic oligo-dG primer is then fhnealed to oligo dC. Tir in turn enables the symthesis of the second svand of cDNA. By this improved. technique, a full length of cDNA corresponding to mRNA (in tun the gene) is obtained. But the efficiency of this method is comparatively lower. Construction of eDNA libraries The complementary DNA molecules can be cloned in cloning vector (plasmid), for creating {DNA libraries. The cDNA insertion ieto the vector ‘should have correct orientation. This is achieved byChapter 9 : GENE LIBRARIES aaa’ ANA Ferrera “isms a Torin ranrase vee &® or oye nA ‘net at) s rm § [ora | anneal t co ms pe * Daslestonded wa Fier Fla, 24 Improved meted for complementary DNA syntesi. the addition of a synthetic linker © the double- stranded cDNA, Ina technique developed by Okayama and Berg, (0982), the mRNA is fist linked to the plasmid Cloning vector and then cDNA synthesis is carried RT-PCR as an alternative to ‘DNA cloning Reverse transcription followed by PCR (RT-PCR) can amplify she mRNA to give cDNA (For details Refer Chapter 8. RT-PCR is very rapid hence cDNA molecules can be obtained in a shor period. Further, even the long length mRNAS can be conveniently used in REPCR, ‘There are some disadvantages also in RE-PCR The DNA polymerase used in REPCR is ertor prone, and even a. ven SERNA (with oth resus. ‘Once a DNA library or 2

Vous aimerez peut-être aussi

• The Subtle Art of Not Giving a F*ck: A Counterintuitive Approach to Living a Good Life

  

  D'Everand

  The Subtle Art of Not Giving a F*ck: A Counterintuitive Approach to Living a Good Life

  Mark Manson

  Évaluation : 4 sur 5 étoiles

  4/5 (5794)
• Kkss - Aaa........ D..sa,............. C....... A....... Standard Plate Count.....

  

  Document5 pages

  Kkss - Aaa........ D..sa,............. C....... A....... Standard Plate Count.....

  Kala King

  Pas encore d'évaluation
• Shoe Dog: A Memoir by the Creator of Nike

  

  D'Everand

  Shoe Dog: A Memoir by the Creator of Nike

  Phil Knight

  Évaluation : 4.5 sur 5 étoiles

  4.5/5 (537)
• • Magazines
  • Podcasts
  • Partition
• Effect of HPMC 4 Cps and Carbopol 974p NF On Release Kinetics of Ciprofloxacin From Kollidon SR Embedded Matrix Tablets

  

  Document9 pages

  Effect of HPMC 4 Cps and Carbopol 974p NF On Release Kinetics of Ciprofloxacin From Kollidon SR Embedded Matrix Tablets

  Kala King

  Pas encore d'évaluation
• Yes Please

  

  D'Everand

  Yes Please

  Amy Poehler

  Évaluation : 4 sur 5 étoiles

  4/5 (1891)
• Industrial Topics

  

  Document2 pages

  Industrial Topics

  Kala King

  Pas encore d'évaluation
• The Yellow House: A Memoir (2019 National Book Award Winner)

  

  D'Everand

  The Yellow House: A Memoir (2019 National Book Award Winner)

  Sarah M. Broom

  Évaluation : 4 sur 5 étoiles

  4/5 (98)
• LAL Vol.13No.2 PDF

  

  Document3 pages

  LAL Vol.13No.2 PDF

  Kala King

  Pas encore d'évaluation
• Hidden Figures: The American Dream and the Untold Story of the Black Women Mathematicians Who Helped Win the Space Race

  

  D'Everand

  Hidden Figures: The American Dream and the Untold Story of the Black Women Mathematicians Who Helped Win the Space Race

  Margot Lee Shetterly

  Évaluation : 4 sur 5 étoiles

  4/5 (895)
• Jkkss - Aaa........ D..sa,............. C....... A....... Standard Plate Count.....

  

  Document5 pages

  Jkkss - Aaa........ D..sa,............. C....... A....... Standard Plate Count.....

  Kala King

  Pas encore d'évaluation
• The Hard Thing About Hard Things: Building a Business When There Are No Easy Answers

  

  D'Everand

  The Hard Thing About Hard Things: Building a Business When There Are No Easy Answers

  Ben Horowitz

  Évaluation : 4.5 sur 5 étoiles

  4.5/5 (344)
• Yadast Ki KamOri 22

  

  Document1 page

  Yadast Ki KamOri 22

  Kala King

  Pas encore d'évaluation
• The Little Book of Hygge: Danish Secrets to Happy Living

  

  D'Everand

  The Little Book of Hygge: Danish Secrets to Happy Living

  Meik Wiking

  Évaluation : 3.5 sur 5 étoiles

  3.5/5 (399)
• ........ D..sa,............. C....... A....... Standard Plate Count....

  

  Document5 pages

  ........ D..sa,............. C....... A....... Standard Plate Count....

  Kala King

  Pas encore d'évaluation
• Grit: The Power of Passion and Perseverance

  

  D'Everand

  Grit: The Power of Passion and Perseverance

  Angela Duckworth

  Évaluation : 4 sur 5 étoiles

  4/5 (588)
• Aaa........ D..sa,............. C....... A....... Standard Plate Count.....

  

  Document5 pages

  Aaa........ D..sa,............. C....... A....... Standard Plate Count.....

  Kala King

  Pas encore d'évaluation
• The Emperor of All Maladies: A Biography of Cancer

  

  D'Everand

  The Emperor of All Maladies: A Biography of Cancer

  Siddhartha Mukherjee

  Évaluation : 4.5 sur 5 étoiles

  4.5/5 (271)
• SPC

  

  Document5 pages

  SPC

  Kala King

  Pas encore d'évaluation
• Devil in the Grove: Thurgood Marshall, the Groveland Boys, and the Dawn of a New America

  

  D'Everand

  Devil in the Grove: Thurgood Marshall, the Groveland Boys, and the Dawn of a New America

  Gilbert King

  Évaluation : 4.5 sur 5 étoiles

  4.5/5 (266)
• Just For Laughs

  

  Document49 pages

  Just For Laughs

  Zareena Carrim

  Pas encore d'évaluation
• Never Split the Difference: Negotiating As If Your Life Depended On It

  

  D'Everand

  Never Split the Difference: Negotiating As If Your Life Depended On It

  Chris Voss

  Évaluation : 4.5 sur 5 étoiles

  4.5/5 (838)
• A........ D..sa,............. C....... A....... Standard Plate Count.....

  

  Document5 pages

  A........ D..sa,............. C....... A....... Standard Plate Count.....

  Kala King

  Pas encore d'évaluation
• A Heartbreaking Work Of Staggering Genius: A Memoir Based on a True Story

  

  D'Everand

  A Heartbreaking Work Of Staggering Genius: A Memoir Based on a True Story

  Dave Eggers

  Évaluation : 3.5 sur 5 étoiles

  3.5/5 (231)
• SPC

  

  Document5 pages

  SPC

  Kala King

  Pas encore d'évaluation
• Principles: Life and Work

  

  D'Everand

  Principles: Life and Work

  Ray Dalio

  Évaluation : 4 sur 5 étoiles

  4/5 (599)
• Aaa........ D..sa,............. C....... A....... Standard Plate Count.....

  

  Document5 pages

  Aaa........ D..sa,............. C....... A....... Standard Plate Count.....

  Kala King

  Pas encore d'évaluation
• On Fire: The (Burning) Case for a Green New Deal

  

  D'Everand

  On Fire: The (Burning) Case for a Green New Deal

  Naomi Klein

  Évaluation : 4 sur 5 étoiles

  4/5 (73)
• ........ D..sa,............. C....... A....... Standard Plate Count.....

  

  Document5 pages

  ........ D..sa,............. C....... A....... Standard Plate Count.....

  Kala King

  Pas encore d'évaluation
• Elon Musk: Tesla, SpaceX, and the Quest for a Fantastic Future

  

  D'Everand

  Elon Musk: Tesla, SpaceX, and the Quest for a Fantastic Future

  Ashlee Vance

  Évaluation : 4.5 sur 5 étoiles

  4.5/5 (474)
• Javed CH

  

  Document7 pages

  Javed CH

  Kala King

  Pas encore d'évaluation
• Team of Rivals: The Political Genius of Abraham Lincoln

  

  D'Everand

  Team of Rivals: The Political Genius of Abraham Lincoln

  Doris Kearns Goodwin

  Évaluation : 4.5 sur 5 étoiles

  4.5/5 (234)
• ..... D..sa,............. C....... A....... Standard Plate Count....

  

  Document5 pages

  ..... D..sa,............. C....... A....... Standard Plate Count....

  Kala King

  Pas encore d'évaluation
• The World Is Flat 3.0: A Brief History of the Twenty-first Century

  

  D'Everand

  The World Is Flat 3.0: A Brief History of the Twenty-first Century

  Thomas L. Friedman

  Évaluation : 3.5 sur 5 étoiles

  3.5/5 (2259)
• ...... D..sa,............. C....... A....... Standard Plate Count....

  

  Document5 pages

  ...... D..sa,............. C....... A....... Standard Plate Count....

  Kala King

  Pas encore d'évaluation
• Angela's Ashes: A Memoir

  

  D'Everand

  Angela's Ashes: A Memoir

  Frank McCourt

  Évaluation : 4.5 sur 5 étoiles

  4.5/5 (440)
• SPC

  

  Document5 pages

  SPC

  Kala King

  Pas encore d'évaluation
• Rise of ISIS: A Threat We Can't Ignore

  

  D'Everand

  Rise of ISIS: A Threat We Can't Ignore

  Jay Sekulow

  Évaluation : 3.5 sur 5 étoiles

  3.5/5 (137)
• D..sa,............. C....... A....... Standard Plate Count....

  

  Document5 pages

  D..sa,............. C....... A....... Standard Plate Count....

  Kala King

  Pas encore d'évaluation
• Steve Jobs

  

  D'Everand

  Steve Jobs

  Walter Isaacson

  Évaluation : 4.5 sur 5 étoiles

  4.5/5 (806)
• ..D..sa,............. C....... A....... Standard Plate Count....

  

  Document5 pages

  ..D..sa,............. C....... A....... Standard Plate Count....

  Kala King

  Pas encore d'évaluation
• Fear: Trump in the White House

  

  D'Everand

  Fear: Trump in the White House

  Bob Woodward

  Évaluation : 3.5 sur 5 étoiles

  3.5/5 (738)
• SPC

  

  Document5 pages

  SPC

  Kala King

  Pas encore d'évaluation
• The Unwinding: An Inner History of the New America

  

  D'Everand

  The Unwinding: An Inner History of the New America

  George Packer

  Évaluation : 4 sur 5 étoiles

  4/5 (45)
• SPC

  

  Document5 pages

  SPC

  Kala King

  Pas encore d'évaluation
• Bad Feminist: Essays

  

  D'Everand

  Bad Feminist: Essays

  Roxane Gay

  Évaluation : 4 sur 5 étoiles

  4/5 (1015)
• SPC

  

  Document5 pages

  SPC

  Kala King

  Pas encore d'évaluation
• John Adams

  

  D'Everand

  John Adams

  David McCullough

  Évaluation : 4.5 sur 5 étoiles

  4.5/5 (2409)
• ... D..sa,............. C....... A....... Standard Plate Count....

  

  Document5 pages

  ... D..sa,............. C....... A....... Standard Plate Count....

  Kala King

  Pas encore d'évaluation
• The Gifts of Imperfection: Let Go of Who You Think You're Supposed to Be and Embrace Who You Are

  

  D'Everand

  The Gifts of Imperfection: Let Go of Who You Think You're Supposed to Be and Embrace Who You Are

  Brené Brown

  Évaluation : 4 sur 5 étoiles

  4/5 (1090)
• SPC

  

  Document5 pages

  SPC

  Kala King

  Pas encore d'évaluation
• The Glass Castle: A Memoir

  

  D'Everand

  The Glass Castle: A Memoir

  Jeannette Walls

  Évaluation : 4.5 sur 5 étoiles

  4.5/5 (1712)
• SPC

  

  Document5 pages

  SPC

  Kala King

  Pas encore d'évaluation
• The Light Between Oceans: A Novel

  

  D'Everand

  The Light Between Oceans: A Novel

  M.L. Stedman

  Évaluation : 4.5 sur 5 étoiles

  4.5/5 (789)
• SPC

  

  Document5 pages

  SPC

  Kala King

  Pas encore d'évaluation
• The Outsider: A Novel

  

  D'Everand

  The Outsider: A Novel

  Stephen King

  Évaluation : 4 sur 5 étoiles

  4/5 (1839)
• SPC

  

  Document5 pages

  SPC

  Kala King

  Pas encore d'évaluation
• The Sympathizer: A Novel (Pulitzer Prize for Fiction)

  

  D'Everand

  The Sympathizer: A Novel (Pulitzer Prize for Fiction)

  Viet Thanh Nguyen

  Évaluation : 4.5 sur 5 étoiles

  4.5/5 (121)
• SPC

  

  Document5 pages

  SPC

  Kala King

  Pas encore d'évaluation
• The Woman in Cabin 10

  

  D'Everand

  The Woman in Cabin 10

  Ruth Ware

  Évaluation : 3.5 sur 5 étoiles

  3.5/5 (2322)
• SPC

  

  Document5 pages

  SPC

  Kala King

  Pas encore d'évaluation
• Brooklyn: A Novel

  

  D'Everand

  Brooklyn: A Novel

  Colm Tóibín

  Évaluation : 3.5 sur 5 étoiles

  3.5/5 (1937)
• Syllabus-for-MTech-Nanoscience - NEW - SRM University

  

  Document10 pages

  Syllabus-for-MTech-Nanoscience - NEW - SRM University

  selvakumar

  Pas encore d'évaluation
• A Man Called Ove: A Novel

  

  D'Everand

  A Man Called Ove: A Novel

  Fredrik Backman

  Évaluation : 4.5 sur 5 étoiles

  4.5/5 (4609)
• Surface Coating: Toxicology Considerations in Nanomedicine

  

  Document3 pages

  Surface Coating: Toxicology Considerations in Nanomedicine

  Muhammad farhan

  Pas encore d'évaluation
• The Perks of Being a Wallflower

  

  D'Everand

  The Perks of Being a Wallflower

  Stephen Chbosky

  Évaluation : 4.5 sur 5 étoiles

  4.5/5 (2102)
• Synthesis of Nano Materials

  

  Document2 pages

  Synthesis of Nano Materials

  Manojkumar A

  Pas encore d'évaluation
• Wolf Hall: A Novel

  

  D'Everand

  Wolf Hall: A Novel

  Hilary Mantel

  Évaluation : 4 sur 5 étoiles

  4/5 (3811)
• Nanoparticle Based Drug Delivery Assignment

  

  Document15 pages

  Nanoparticle Based Drug Delivery Assignment

  showrav

  100% (1)
• Little Women

  

  D'Everand

  Little Women

  Louisa May Alcott

  Évaluation : 4 sur 5 étoiles

  4/5 (104)
• Nanotechnology and Its Future Applications in Medicine, Food, Computational Systems and Energy

  

  Document2 pages

  Nanotechnology and Its Future Applications in Medicine, Food, Computational Systems and Energy

  Faiz Rasool

  Pas encore d'évaluation
• A Tree Grows in Brooklyn

  

  D'Everand

  A Tree Grows in Brooklyn

  Betty Smith

  Évaluation : 4.5 sur 5 étoiles

  4.5/5 (1929)
• Nanotechnology Regulatory Needs

  

  Document38 pages

  Nanotechnology Regulatory Needs

  daiceman

  Pas encore d'évaluation
• Manhattan Beach: A Novel

  

  D'Everand

  Manhattan Beach: A Novel

  Jennifer Egan

  Évaluation : 3.5 sur 5 étoiles

  3.5/5 (792)
• CHAPTER 2 Classification of Nanomaterials

  

  Document45 pages

  CHAPTER 2 Classification of Nanomaterials

  Farah Hanani Zulkifli

  Pas encore d'évaluation
• The Art of Racing in the Rain: A Novel

  

  D'Everand

  The Art of Racing in the Rain: A Novel

  Garth Stein

  Évaluation : 4 sur 5 étoiles

  4/5 (4200)
• MARGALLO - Asynchronous 11 Task 11 (15 Nanotechnology Products)

  

  Document5 pages

  MARGALLO - Asynchronous 11 Task 11 (15 Nanotechnology Products)

  Sam Margallo

  Pas encore d'évaluation
• Sing, Unburied, Sing: A Novel

  

  D'Everand

  Sing, Unburied, Sing: A Novel

  Jesmyn Ward

  Évaluation : 4 sur 5 étoiles

  4/5 (1103)
• Nanolithography - Processing Methods PDF

  

  Document10 pages

  Nanolithography - Processing Methods PDF

  Nilesh Bhardwaj

  Pas encore d'évaluation
• Her Body and Other Parties: Stories

  

  D'Everand

  Her Body and Other Parties: Stories

  Carmen Maria Machado

  Évaluation : 4 sur 5 étoiles

  4/5 (821)
• EMAMOS Store Details 9 Mega Gloryoung International Revised 1

  

  Document13 pages

  EMAMOS Store Details 9 Mega Gloryoung International Revised 1

  Mark Iom

  Pas encore d'évaluation
• The Constant Gardener: A Novel

  

  D'Everand

  The Constant Gardener: A Novel

  John le Carré

  Évaluation : 3.5 sur 5 étoiles

  3.5/5 (104)
• The Nano World: Republic of The Philippines Nabua, Camarines Sur

  

  Document4 pages

  The Nano World: Republic of The Philippines Nabua, Camarines Sur

  Raymon Villapando Bardinas

  Pas encore d'évaluation
• Techně Logos: Law Formulated by Isaac Asimov Back in 1940's

  

  Document8 pages

  Techně Logos: Law Formulated by Isaac Asimov Back in 1940's

  Sofia Gadoy

  Pas encore d'évaluation
• 2018년 중소기업 전략기술로드맵 09 첨단소재★

  

  Document255 pages

  2018년 중소기업 전략기술로드맵 09 첨단소재★

  Sean Choi

  Pas encore d'évaluation
• Nanotechnology in Dentistry - Soon To Be Called Nanodontics

  

  Document12 pages

  Nanotechnology in Dentistry - Soon To Be Called Nanodontics

  IJAR JOURNAL

  100% (1)
• Nano All

  

  Document183 pages

  Nano All

  venu

  Pas encore d'évaluation
• Seminar On Nanorobotics

  

  Document23 pages

  Seminar On Nanorobotics

  Prachi Kulkarni

  Pas encore d'évaluation
• Gao Yuqing PHD Final 151117

  

  Document146 pages

  Gao Yuqing PHD Final 151117

  Cody Lee

  Pas encore d'évaluation
• Foundations of Nanotechnology - Three Volume Set - Foundations of Nanotechnology, Volume Three - Mechanics of Carbon Nanotubes (PDFDrive)

  

  Document290 pages

  Foundations of Nanotechnology - Three Volume Set - Foundations of Nanotechnology, Volume Three - Mechanics of Carbon Nanotubes (PDFDrive)

  Aruesirioghene Justice

  Pas encore d'évaluation
• Nano Assignment #1

  

  Document16 pages

  Nano Assignment #1

  Aftab Ahmad

  Pas encore d'évaluation
• Membran Bab 1

  

  Document23 pages

  Membran Bab 1

  Kiki Rezki Lestari

  Pas encore d'évaluation
• Aqua Nano Laundry

  

  Document5 pages

  Aqua Nano Laundry

  firinaluvina

  0% (1)
• Solar Panel Cleaning Ashis

  

  Document20 pages

  Solar Panel Cleaning Ashis

  Deepak Kumar Singh

  Pas encore d'évaluation
• Nano

  

  Document10 pages

  Nano

  Arunakirinathan Anapayan

  Pas encore d'évaluation
• Name: Omkar Anil Kirdat Topic: Nanowires Uid No.: 201950113 Roll No.: 13

  

  Document11 pages

  Name: Omkar Anil Kirdat Topic: Nanowires Uid No.: 201950113 Roll No.: 13

  Omkar Kirdat

  Pas encore d'évaluation
• Applications of Bionanotechnology in Nanomedicine: A Theoretical Analysis

  

  Document6 pages

  Applications of Bionanotechnology in Nanomedicine: A Theoretical Analysis

  Philip Kpae

  Pas encore d'évaluation
• L.) UNIT - 12 - NANOTECH PDF

  

  Document28 pages

  L.) UNIT - 12 - NANOTECH PDF

  Mummy Hartini

  Pas encore d'évaluation
• Nanostructures and Nanomaterials

  

  Document2 pages

  Nanostructures and Nanomaterials

  Santosh Mysore

  Pas encore d'évaluation
• Nanotechnology For Water Treatment: K.M.Hundekar 1SI10HCE07

  

  Document20 pages

  Nanotechnology For Water Treatment: K.M.Hundekar 1SI10HCE07

  CHIEF VISHAAL 45

  Pas encore d'évaluation
• Nanocomposites Materials Definitions Typ 767a08f9

  

  Document7 pages

  Nanocomposites Materials Definitions Typ 767a08f9

  Ahmed Tantawy

  Pas encore d'évaluation
• Why The Future Does Not Need Us: Intended Learning Outcomes

  

  Document2 pages

  Why The Future Does Not Need Us: Intended Learning Outcomes

  Norsaidin Tungkag

  Pas encore d'évaluation