GENETIC Engineering SMITA RASTOGI Department of Biotechnology Delhi Technological University Delhi NEELAM PATHAK Department of Biotechnology Integral University Lucknow © Oxford University Press Contents Preface v List of Abbreviations x PART I: FUNDAMENTALS OF GENETIC ENGINEERING 1. An Introduction to Gene Cloning 2 3.8 Applications of Restriction 1.1 Introduction 2 Endonucleases 91 1.2 Cloning Vectors 7 4. Chemical Synthesis of Oligonucleotides 102 1.3 Steps Involved in Gene Cloning 10 4.1 Introduction 102 1.4 Subcloning 40 4.2 Solution-phase vs. Solid-phase Synthesis 102 1.5 Advantages of Gene or cDNA Cloning 40 4.3 Historical Perspective of 2. Enzymes Used in Genetic Engineering 43 Chemical Synthesis of DNA 103 2.1 Introduction 43 4.4 Conventional Solid-phase 2.2 Restriction Endonuclease 43 Synthesis: Phosphoramidite Method 105 2.3 DNA Polymerase (DNA 4.5 DNA Synthesizers Nucleotidyl-transferase; EC 2.7.7.7) 43 (Automated Solid-Phase Synthesis) 116 2.4 Reverse Transcriptase (RNA-directed DNA 4.6 Systematic Evolution of Ligands Polymerase, EC 2.7.7.49) 49 by Exponential Enrichment (SELEX) 118 2.5 RNA Polymerase (RNA Nucleotidyl 4.7 DNA Chips 118 Transferase; EC 2.7.7.6) 50 5. Synthesis of Complementary DNA 131 2.6 Alkaline Phosphatase (Orthophosphoric-monoester 5.1 Introduction 131 Phosphohydrolase; EC 3.1.3.1) 53 5.2 Enrichment and Purification of mRNA 131 2.7 Polynucleotide Kinase (PNK) 5.3 cDNA Synthesis 143 (ATP: 5¢-Dephosphopolynucleotide 5¢-Phosphotransferase; EC 2.7.1.78) 53 6. Techniques for Nucleic Acid Analysis and Size 2.8 DNA Ligase Fractionation 164 (Polydeoxyribonucleotide Synthetase) 55 6.1 Introduction 164 2.9 Deoxyribonuclease (DNase) 56 6.2 Gel Electrophoresis 164 2.10 Ribonuclease (RNase) 63 6.3 Elution of DNA Fragments from Gel 195 2.11 Phosphodiesterase I (EC 3.1.4.41) 64 6.4 Gel Filtration Chromatography 198 2.12 b-Agarase (EC 3.2.1.81) 65 6.5 Density Gradient Centrifugation 201 2.13 UracilDNA Glycosylase (EC 3.2.2.2.3) 65 7. Polymerase Chain Reaction 207 2.14 Proteinase K (EC 3.4.21.64) 65 7.1 Introduction 207 2.15 Lysozyme (EC 3.2.1.17) 66 7.2 Setting up PCR reaction 207 2.16 Topoisomerase 66 7.3 Thermostable DNA Polymerases 208 3. Restriction Endonucleases 68 7.4 Primer Designing 208 3.1 Introduction 68 7.5 Cycle Number 210 3.2 Host Controlled Restriction and 7.6 PCR Product Yield 211 Modification (R-M) System 68 7.7 Verification of PCR Product 211 3.3 Nomenclature of Restriction Endonucleases 70 7.8 Factors Affecting PCR Amplification 211 3.4 Types of Restriction Endonucleases 70 7.9 Variants of PCR 212 3.5 Recognition Sites 79 7.10 Isothermal Techniques of DNA 3.6 Cleavage by Restriction Endonucleases 81 Amplification 224 3.7 Variants of Restriction Endonucleases 87 7.11 Applications of PCR 226 © Oxford University Press GE Prelims (6th Proof).p65 7 7/3/10, 7:11 PM viii Contents PART II : VECTORS USED IN GENE CLONING 8. Plasmids 236 10.2 Biology of Filamentous Bacteriophages 322 8.1 Introduction 236 10.3 M13 Bacteriophage as Cloning Vector 329 8.2 Definitions of Plasmid 237 10.4 Cloning in M13 Vectors 332 8.3 Why is Plasmid Not Considered Genome? 238 11. Yeast Cloning Vectors 337 8.4 Plasmid vs. Virus 238 11.1 Introduction 337 8.5 Plasmid Size Range 238 11.2 Why is Yeast System Required? 337 8.6 Shapes of Plasmids: Covalently 11.3 Nutritional Auxotrophic Complementation Closed Circular and Linear plasmids 238 for Recombinant Selection 339 8.7 Replication of Plasmids 239 11.4 Transformation of Yeast Cells 342 8.8 Plasmid Host Range 242 11.5 Yeast 2-mm Plasmid 342 8.9 Stable Maintenance of Plasmids 243 11.6 Yeast Cloning Vectors 344 8.10 Control of Plasmid Copy Number 243 12. Vectors Other Than E. coli Plasmids, 8.11 Plasmid Incompatibility 247 l Bacteriophage, and M13 361 8.12 Plasmid Classification 248 8.13 Plasmid Purification 258 12.1 Introduction 361 8.15 Plasmids as Vectors in Genetic Engineering 260 12.2 Chimeric Vectors 361 12.3 Gram Negative Bacteria Other Than 9. l Bacteriophage 271 E. coli as Cloning Vectors 373 9.1 Introduction 271 12.4 Gram Positive Bacteria as Cloning 9.2 Biology of l Bacteriophage 271 Vectors 381 9.3 l Bacteriophage Growth and Preparation 12.5 Plant and Animal Viral Vectors 383 of l DNA 295 12.6 P1 Phage as Vector 395 9.4 Methods of Introduction of l DNA into 12.7 Fungal Systems Other Than Yeast 395 Bacterial Cells 298 12.8 Artificial Chromosomes 397 9.5 l Bacteriophage as Cloning Vehicle 300 12.9 Shuttle Vectors 398 10. M13 Bacteriophage 322 12.10 Expression Vectors 398 10.1 Introduction 322 12.11 Advanced Gene Trapping Vectors 410 PART III : GENERATION AND SCREENING OF RECOMBINANTS 13. Joining of DNA Fragments 414 15. Construction of Genomic and cDNA Libraries 469 13.1 Introduction 414 15.1 Introduction 469 13.2 Ligation of DNA Fragments using DNA 15.2 Genomic Library 469 Ligase 414 15.3 cDNA Library 476 13.3 Ligation Using Homopolymer Tailing 421 15.4 PCR as an Alternative to Library 13.4 Increasing Versatility and Efficiency of Construction 483 Ligation by Modification of the Ends of 16. Techniques for Selection, Screening, and Restriction Fragments 423 Characterization of Transformants 487 14. Introduction of DNA into Host Cells 430 16.1 Introduction 487 14.1 Introduction 430 16.2 Selectable Marker Genes 487 14.2 Introduction of DNA into Bacterial 16.3 Reporter Genes 490 Cells 435 16.4 Screening of Clone(s) of Interest 494 14.3 Introduction of DNA into Yeast Cells 441 16.5 Nucleic Acid Blotting and Hybridization 495 14.4 Genetic Transformation of Plants 441 16.6 Protein Structure/Function-based 14.5 Introduction of DNA into Insects 459 Techniques 518 © Oxford University Press GE Prelims (6th Proof).p65 8 7/3/10, 7:11 PM Contents ix PART IV: APPLIED GENETIC ENGINEERING 17. Other Techniques for Genetic Manipulation 528 20. Applications of Cloning and Gene Transfer 17.1 Introduction 528 Technology 613 17.2 In vitro Mutagenesis 528 20.1 Introduction 613 17.3 Gene Manipulation by Recombination 541 20.2 Applications of Recombinant 17.4 Techniques Based on Gene Silencing 545 Microorganisms 613 18. Molecular Markers 554 20.3 Applications of Transgenic Plant 18.1 Introduction 554 Technology 629 18.2 Biochemical Markers 554 20.4 Applications of Animal Cloning and 18.3 Molecular Markers 555 Transgenic Technology 657 18.4 Restriction Fragment Length 21. Safety Regulations Related to Genetic Polymorphism 556 Engineering 679 18.5 Random Amplified Polymorphic DNA 558 21.1 Introduction 679 18.6 Amplified Fragment Length 21.2 National Regulatory Mechanism Polymorphism 561 for Implementation of Biosafety 18.7 Microsatellite or Short Tandem Guidelines for Handling GMOs 679 Repeats 564 21.3 Salient Features of Recombinant DNA 18.8 Single Strand Conformation Biosafety Guidelines, Revised Guidelines Polymorphism 567 for Research in Transgenic Plants and Risk 18.9 Single Nucleotide Polymorphism 568 Assessment 683 18.10 Map-based Cloning 570 21.4 Regulations for GM Plants: Bt Cotton 18.11 Marker-assisted Selection 570 in Indiaa Case Study 689 19. Techniques Used in Genomics and Proteomics 573 21.5 Regulations Related to Stem Cell Research 19.1 Introduction 537 and Human Cloning 697 19.2 Techniques Used in Genomics 573 Glossary 701 19.3 Techniques Used in Proteomics 592 Index 725 © Oxford University Press GE Prelims (6th Proof).p65 9 7/3/10, 7:11 PM An Introduction to 1 Gene Cloning Key Concepts In this chapter we will learn the following: · Cloning vectors · Subcloning · Steps involved in gene cloning · Advantages of gene or cDNA cloning 1.1 INTRODUCTION for gene manipulation in vitro came with the discovery of natural gene transfer capability of viruses and Agrobacterium Gene cloning, developed in the early 1970s, was a signifi- tumefaciens into hosts and development of techniques for cant breakthrough in the field of molecular biology. The tech- genetic transformation of E. coli. Another area of research nique was pioneered by Paul Berg, Herbert Boyer, and Stanley that laid the foundation of gene cloning was renaturation Cohen and was the fruit of several decades of basic research analysis that led to the discovery of base pairing characteris- on nucleic acids (Exhibits 1.1 and 1.2). Further contributing tics of two complementary sequences (i.e., hybridization), factors for the development of gene cloning technique were indicating that complementary DNA or RNA probes can be critical understanding of E. coli genetics, information about used to detect specific nucleotide sequences. nucleic acid enzymology (i.e., having enzymes that cut, join, The technique of gene cloning (also called molecular clon- and replicate DNA or reverse transcribe RNA), availability ing) essentially involves the insertion of target DNA (also of techniques for monitoring the cutting and joining reac- called foreign DNA/passenger DNA/exogenous DNA/insert tions, and information about bacterial plasmids. The impetus DNA/DNA fragment/gene of interest) into a cell through a Exhibit 1.1 Deoxyribonucleic acid (DNA) DNA is a high molecular weight biopolymer comprising of carbonyl oxygen at C2 position (carbon atom at position 2) of mononucleotides as their repeating units, which are joined by pyrimidine exist in anti form. Besides, the major nitrogenous bases, 3’ ® 5’ phosphodiester bonds. Each mononucleotide unit of DNA some minor bases called modified nitrogenous bases also occur in consists of purine and pyrimidine nitrogenous bases, phosphorus, polynucleotide structures, for example, 5,6-dihydrouracil, and a pentose sugar 2’-deoxy-D-ribose (Figure A).