Techniques in Genetic Engineering

LIFE SCIENCES Kurnaz Techniques in Techniques in GENETIC ENGINEERING in Techniques GENETIC 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 ENGINEERING 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. GENETIC 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. ENGINEERING 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. K24275 Işıl Aksan Kurnaz 6000 Broken Sound Parkway, NW Suite 300, Boca Raton, FL 33487 711 Third Avenue New York, NY 10017 an informa business 2 Park Square, Milton Park www.crcpress.com Abingdon, Oxon OX14 4RN, UK www.crcpress.com Techniques in GENETIC ENGINEERING Techniques in GENETIC ENGINEERING Işıl Aksan Kurnaz Boca Raton London New York CRC Press is an imprint of the Taylor & Francis Group, an informa business 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 No claim to original U.S. Government works Version Date: 20150409 International Standard Book Number-13: 978-1-4822-6090-8 (eBook - PDF) This book contains information obtained from authentic and highly regarded sources. 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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 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 ix 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

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