Techniques in Genetic Engineering

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Techniques in

GENETIC

ENGINEERING

Işıl Aksan Kurnaz

Techniques in

GENETIC ENGINEERING

Kurnaz Techniques in GENETIC ENGINEERING

Although designed for undergraduates with an interest in molecular biology,

biotechnology, and bioengineering, this book—Techniques in Genetic

Engineering—IS NOT a laboratory manual; nor is it a textbook on molecular

biology or biochemistry. There is some basic information in the appendices about

core concepts such as DNA, RNA, protein, genes, and genomes; however, in

general it is assumed that the reader has a background on these key issues.

Techniques in Genetic Engineering briefly introduces some common genetic

engineering techniques and focuses on how to approach different real-life

problems using a combination of these key issues. Although not an exhaustive

review of these techniques, basic information includes core concepts such as

DNA, RNA, protein, genes, and genomes. It is assumed that the reader has a

background on these key issues. The book provides sufficient background and

future perspectives for the readers to develop their own experimental strategies

and innovations.

This easy-to-follow book presents not only the theoretical background of

molecular techniques, but also provides case study examples, with some

sample solutions. The book covers basic molecular cloning procedures; genetic

modification of cells, including stem cells; as well as multicellular organisms,

using problem-based case study examples.

Techniques in

GENETIC

ENGINEERING

Boca Raton London New York

CRC Press is an imprint of the

Taylor & Francis Group, an informa business

Techniques in

GENETIC

ENGINEERING

Işıl Aksan Kurnaz

CRC Press

Taylor & Francis Group

6000 Broken Sound Parkway NW, Suite 300

Boca Raton, FL 33487-2742

© 2015 by Taylor & Francis Group, LLC

CRC Press is an imprint of Taylor & Francis Group, an Informa business

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Version Date: 20150409

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To my husband and partner in life,

my two adorable kids,

my parents and my family,

my devoted assistants,

and all my students …

We must not forget that when radium was discov￾ered no one knew that it would prove useful in

hospitals. The work was done of pure science. And

this is a proof that scientific work must not be con￾sidered from the point of view of the direct useful￾ness of it. It must be done for itself, for the beauty

of science, and then there is always the chance that

a scientific discovery may become like the radium a

benefit for humanity.

Marie Curie (1867–1934)

Lecture at Vassar College, May 14, 1921

ix

Contents

Preface..........................................................................xv

Acknowledgments ..................................................... xvii

Abbreviations and Acronyms ..................................... xix

1 Introduction to Genetic Engineering.......................1

2 Tools of Genetic Engineering ..................................7

2.1 Restriction Endonucleases.......................................... 8

2.1.1 Type I Endonucleases...................................... 9

2.1.2 Type II Endonucleases .................................... 9

2.1.3 Type IIs Endonucleases..................................12

2.1.4 Type III Endonucleases ..................................12

2.1.5 Type IV Endonucleases ..................................12

2.1.6 Isoschizomers and Neoschizomers................13

2.1.7 Star Activity.....................................................14

2.1.8 Restriction Mapping........................................15

2.1.9 Restriction Fragment Length Polymorphism...15

2.2 Vectors .......................................................................19

2.2.1 Plasmids ..........................................................19

2.2.2 Phage Vectors .................................................26

2.2.3 Cosmids and Phagemids ................................31

2.2.4 Specialist Vectors ............................................35

2.2.4.1 Bacterial Artificial Chromosomes .....35

2.2.4.2 Yeast Artificial Chromosomes...........36

2.2.4.3 Expression Vectors ............................37

x ◾ Contents

2.3 Modifying Enzymes...................................................39

2.3.1 Polymerases ....................................................41

2.3.2 Ligases.............................................................43

2.3.3 Alkaline Phosphatases....................................47

2.3.4 Recombinases .................................................47

2.4 Basic Principles of Cloning ...................................... 48

2.4.1 Bacterial Transformation ............................... 48

2.4.2 Screening for Recombinants ..........................50

2.5 Problem Session ........................................................55

3 DNA Libraries ........................................................61

Introduction .......................................................................61

3.1 Genomic DNA Libraries ............................................62

3.2 cDNA Libraries ......................................................... 64

3.3 Library Screening ......................................................67

3.4 Monitoring Transcription...........................................67

3.4.1 RT-PCR ........................................................... 68

3.4.2 Northern Blotting ...........................................71

3.4.3 Nuclease Protection Assay .............................73

3.4.4 Microarray Analysis ........................................75

3.5 Problem Session ....................................................... 77

4 Protein Production and Purification .....................81

4.1 Expression Vectors and Recombinant Protein

Expressions................................................................82

4.2 In Vitro Transcription and Translation......................83

4.3 Bacterial Expression of Proteins ...............................85

4.4 Expressions in Yeast................................................. 86

4.5 Expressions in Insect Cells ...................................... 88

4.6 Expressions in Plant Cells ........................................ 90

4.7 Expressions in Mammalian Cells ..............................91

4.8 Purification of Proteins..............................................91

4.8.1 Affinity Purification by Nickel Columns........92

4.8.2 Affinity Purification Using Monoclonal

and Polyclonal Antibodies..............................94

Contents ◾ xi

4.8.3 Monitoring Expressions in Cells ....................97

4.8.4 Creating Fusion Proteins: Green

Fluorescent Proteins .....................................100

4.9 Post-Translational Modifications of Proteins...........101

4.10 Problem Session ......................................................104

5 Mutagenesis .........................................................109

5.1 Mutagenesis .............................................................109

5.2 Deletion Studies.......................................................110

5.3 Site-Directed Mutagenesis .......................................121

5.4 Random Mutagenesis ..............................................123

5.5 Directed Evolution, Protein Engineering, and

Enzyme Engineering ...............................................124

5.6 Problem Session ......................................................126

6 Protein–Protein Interactions ...............................129

Introduction .....................................................................129

6.1 GST Pull-Down-Based Interaction Assay ...............130

6.2 Co-Immunoprecipitation .........................................132

6.3 The Yeast Two-Hybrid Assay..................................132

6.4 Fluorescence Resonance Energy Transfer ..............139

6.5 Problem Session ......................................................141

7 Cell Culture..........................................................145

Introduction ..................................................................... 145

7.1 Genetic Manipulation of Cells ................................148

7.1.1 Electrical Methods ........................................149

7.1.2 Mechanical Methods.....................................150

7.1.3 Chemical Methods ........................................150

7.1.4 Viral Methods ............................................... 151

7.1.5 Laser Methods............................................... 157

7.2 Reporter Genes........................................................ 157

7.3 Types of Transfection.............................................. 161

7.3.1 Transient Transfection .................................. 161

7.3.2 Stable Transfection........................................ 161

xii ◾ Contents

7.3.3 Recombination and Integration into the

Genome ........................................................162

7.3.3.1 Homologous Recombination ..........162

7.3.3.2 Site-Specific Recombination............163

7.4 Level of Expression .................................................165

7.4.1 Constitutive Expression ................................165

7.4.2 Inducible Expression ....................................165

7.5 Problem Session ......................................................166

8 Genetic Manipulation of Stem Cells and Animals ...169

8.1 Stem Cell Technology and Knockout Cells ............170

8.1.1 Genetic Manipulation of Embryonic Stem

Cells ..............................................................172

8.1.2 Induced Pluripotent Stem Cells (iPSCs) ....... 175

8.2 Transgenic Animals .................................................177

8.3 RNA Interference and MicroRNAs ..........................183

8.4 Animal Cloning .......................................................186

8.5 Pharm Animals ........................................................188

8.6 Gene Therapy..........................................................189

8.7 Genome Editing.......................................................192

8.8 Problem Session ......................................................195

9 Genetic Manipulation of Plants ...........................197

9.1 Monocotyledons, Dicotyledons, and Commercial

Crops........................................................................199

9.2 Plant Manipulation Methods ...................................202

9.2.1 Plant Cell and Tissue Culture.......................202

9.2.2 The Gene Gun..............................................203

9.2.3 Protoplasts ....................................................204

9.2.4 Agrobacterium ..............................................204

9.2.5 Plant Expression and Reporter Vectors........206

9.3 Future Trends in Transgenic Plants ........................209

9.4 Problem Session ......................................................210

10 Today and the Future...........................................213

10.1 Bioinformatics and the Omics Age.........................214

10.2 Synthetic Biology and Unnatural Amino Acids...... 217

Contents ◾ xiii

10.3 Optogenetics............................................................220

10.4 What Is Next?...........................................................222

10.5 Problem Session ......................................................223

References ..................................................................225

Glossary......................................................................237

Appendix A: DNA Techniques ....................................257

A.1 DNA Gel Electrophoresis ........................................257

A.2 Nucleic Acid Blotting...............................................259

A.3 Polymerase Chain Reaction.....................................261

A.4 Real-Time PCR .........................................................263

A.5 DNA Sequencing .....................................................264

A.6 Next Generation Sequencing ..................................265

Appendix B: Protein Techniques................................269

B.1 SDS-PAGE ................................................................269

B.2 Western Blotting......................................................271

B.3 2D Gel Electrophoresis and Proteomics.................273

B.4 Immunofluorescence and Immunohistochemistry...275

Appendix C: Supplement Information for End-of￾Chapter Questions..................................................279

C.1 Nucleotides and Nucleic Acids................................279

C.2 Genetic Coding Tables ............................................279

C.3 Amino Acids ............................................................283

C.4 Calculations Regarding Nucleic Acids, Amino

Acids, and Proteins..................................................283

C.4.1 Spectrophotometric Measurements..............283

C.4.2 Average Molecular Weights ..........................285

C.4.3 Conversions of Molecular Weights for

Protein and DNA ..........................................285

C.5 Compatible Overhangs............................................285

C.6 Genotypes of Frequently Used Laboratory

Strains of Bacteria and Yeast ..................................287

C.7 Methylation Sensitivities of Common Restriction

Enzymes...................................................................287