Gene Cloning » (4th Edition)

Dr Julia Lodge is Assistant Professor (Lecturer) at the School of Biosciences, University of Birmingham, UK and teaches gene cloning, genetic engineering, genomics. Her main research interest is bacterial infectious diseases.

Dr Pete Lund is Associate Professor (Senior Lecturer) at the School of Biosciences, University of Birmingham, UK and teaches cellular and molecular biology, genetics, protein structure and function. His research focuses on molecular chaperones and stress responses.

Dr Steve Minchin is Associate Professor (Senior Lecturer) at the School of Biosciences, University of Birmingham, UK and teaches genetics, gene expression, genomics. His main area of research is the regulation of gene expression.

The ability to successfully clone genes underlies the majority of our knowledge in molecular and cellular biology. Gene Cloning introduces the diverse array of techniques available to clone genes and how they can be used effectively both in the research laboratory, to gain knowledge about the gene, and for use in biotechnology, medicine, the pharmaceutical industry, and agriculture. It shows how cloning genes is an integral part of genomics and underlines its relevance in the post-genomic age, as a tool required to test predictions of gene regulation and function made through bioinformatics. Applications of gene cloning in medicine, both for diagnosis and treatment, and in the pharmaceutical industry and agriculture, are also covered in the book.

Gene Cloning takes a fresh approach to teaching molecular and cellular biology and will be a valuable resource to both undergraduates and lecturers of biological and biomedical science courses.

Doody Review Services

Reviewer:Eugene A Davidson, PhD(Georgetown University School of Medicine)
Description:This book, for a course in molecular biology, assumes that the student will have been introduced to basic biochemistry, including aspects of information flow, DNA structure, etc.
Purpose:The goal is to provide a text for a senior level course in gene cloning/molecular biology. This worthwhile objective is well achieved.
Audience:The target audience is really the instructor of the course where the material is taught. Students taking the course are the secondary audience. Either way, the authors have chosen well. Although not household names in the scientific arena, the authors clearly have taught this material and know how it should be done.
Features:An initial chapter on the organization of genomes is followed by material on gene cloning, use of DNA libraries, sequencing, bioinformatics, protein expression, and the utilization of transgenic organisms. Each of the 12 main chapters has illustrative examples and sample problems as well as a short supplemental reading list that highlights key contributions from the primary literature. The flow of material is logical and the presentation clear. In addition, the information is current and largely, if not completely, error free. Instructors seeking a text for a course in molecular biology would be wise to consider this one. The authors are to be commended for a readable and well organized presentation.
Assessment:This is a strong effort. The presentation is logical, the level correct. Were I to teach a course in this area, this book would be high on my list.

Chapter 1 Introduction

1.1 The Beginning of Gene Cloning 1

1.2 How To Use This Book 3

1.3 What You Need To Know Before You Read This Book 5

1.4 A Request From the Authors 5

Further Reading 6

Chapter 2 Genome Organization

2.1 Introduction 7

2.2 The C-value Paradox 8

2.3 The Human Genome 9

2.4 Genomes of Other Eukaryotes 19

2.5 Bacterial Genomes 24

2.6 Plasmids 25

2.7 Viral Genomes 26

2.8 GC Content 27

2.9 Physical Characteristics of Eukaryotic Chromosomes 28

2.10 Karyotype 28

2.11 Euchromatin and Heterochromatin 30

2.12 CpG Islands 31

Questions and Answers 32

Further Reading 34

Chapter 3 Key Tools for Gene Cloning

3.1 Introduction 35

3.2 Vectors 36

3.3 Restriction Enzymes 38

3.4 DNA Ligase 40

3.5 Transformation 42

3.6 Purification of Plasmid DNA 45

3.7 More Restriction Enzymes 47

3.8 Alkaline Phosphatase 51

3.9 More About Vectors 53

3.10 Analyzing Cloned DNA by Restriction Mapping 58

3.11 Measuring the Size of DNA Fragments 59

3.12 The Polymerase Chain Reaction and Its Use in Gene Cloning 64

3.13 How Does PCR Work? 67

3.14 Designing PCR Primers 72

3.15 The PCR Reaction 73

3.16 Uses for PCR Products 74

3.17 Cloning PCR Products 74

3.18 Real-time PCR for Quantification of DNA 76

3.19 Advantages and Limitations of PCR 76

Questions and Answers 78

Further Reading 83

Chapter 4 Gene Identification and DNA Libraries

4.1 The Problem 85

4.2 Genomic Library 87

4.3 Constructing a Genomic Library 87

4.4 How Many Clones? 89

4.5 Some DNA Fragments are Under-represented in Genomic Libraries 90

4.6 Using Partial Digests to Make a Genomic Library 90

4.7 Storage of Genomic Libraries 92

4.8 Advantages andDisadvantages of Genomic Libraries 92

4.9 Cloning Vectors for Gene Libraries 93

4.10 Vectors Derived from Bacteriophage [lambda] 93

4.11 Packing Bacteriophage [lambda] In Vitro 95

4.12 Cloning with Bacteriophage [lambda] 97

4.13 Calculating the Titer of your Library 98

4.14 Cosmid Libraries 98

4.15 Making a Cosmid Library 99

4.16 YAC and BAC Vectors 100

4.17 cDNA Libraries 101

4.18 Making a cDNA Library 103

4.19 Cloning the cDNA Product 105

4.20 Expressed Sequence Tags 108

4.21 What are the Disadvantages of a cDNA Library? 108

Questions and Answers 109

Further Reading 116

Chapter 5 Screening DNA Libraries

5.1 The Problem 117

5.2 Screening Methods Based on Gene Expression 118

5.3 Complementation 119

5.4 Immunological Screening of Expression Libraries 120

5.5 Screening Methods Based on Detecting a DNA Sequence 123

5.6 Oligonucleotide Probes 124

5.7 Cloned DNA Fragments as Probes 127

5.8 Colony and Plaque Hybridization 127

5.9 Differential Screening 132

5.10 Using PCR to Screen a Library 133

Questions and Answers 135

Further Reading 140

Chapter 6 Further Routes to Gene Identification

6.1 How Do We Get From Phenotype to Gene: a Fundamental Problem in Gene Cloning 141

6.2 Gene Tagging: A Method That Both Mutates and Marks Genes 142

6.3 A Simple Example of Transposon Tagging in Bacteria: Cloning Adherence Genes from Pseudomonas 149

6.4 Signature-tagged Mutagenesis: Cloning Bacterial Genes with "Difficult" Phenotypes 152

6.5 Gene Tagging in Higher Eukaryotes: Resistance Genes in Plants 156

6.6 Positional Cloning: Using Maps to Track Down Genes 159

6.7 Identification of a Linked Marker 161

6.8 Moving From the Marker Towards the Gene of Interest 161

6.9 Identifying the Gene of Interest 166

6.10 Cloning of the CF Gene: A Case Study 168

Questions and Answers 169

Further Reading 171

Chapter 7 Sequencing DNA

7.1 Introduction 173

7.2 Overview of Sequencing 174

7.3 Sanger Sequencing 175

7.4 The Sanger Sequencing Protocol Requires a Single-stranded DNA Template 179

7.5 Modifications of the Original Sanger Protocol 181

7.6 Strategies for Sequencing a DNA Fragment 182

7.7 High-throughput Sequencing Protocols 184

7.8 The Modern Sequencing Protocol 185

7.9 Genome Sequencing 188

7.10 High-throughput Pyrosequencing 197

7.11 The Importance of DNA Sequencing 202

Questions and Answers 203

Further Reading 205

Chapter 8 Bioinformatics

8.1 Introduction 207

8.2 What Does a Gene Look Like? 208

8.3 Identifying Eukaryotic Genes 214

8.4 Sequence Comparisons 217

8.5 Pair-wise Comparisons 217

8.6 Identity and Similarity 220

8.7 Is the Alignment Significant? 222

8.8 What Can Alignments Tell Us About the Biology of the Sequences Being Compared? 224

8.9 Similarity Searches 224

8.10 Fasta 226

8.11 BLAST 228

8.12 What Can Similarity Searches Tell Us About the Biology of the Sequences Being Compared? 230

8.13 Multiple Sequence Alignments 233

8.14 What Can Multiple Sequence Alignments Tell Us About the Structure and Function of Proteins? 234

8.15 Consensus Patterns and Sequence Motifs 235

8.16 Investigating the Three-dimensional Structures of Biological Molecules 237

8.17 Using Sequence Alignments to Create a Phylogenetic Tree 239

Questions and Answers 242

Further Reading 246

Chapter 9 Production of Proteins from Cloned Genes

9.1 Why Express Proteins? 249

9.2 Requirements for Protein Production from Cloned Genes 252

9.3 The Use of E. coli as a Host Organism for Protein Production 252

9.4 Some Problems in Obtaining High Level Production of Proteins in E. coli 260

9.5 Beyond E. coli: Protein Expression in Eukaryotic Systems 265

9.6 A Final Word About Protein Purification 274

Questions and Answers 275

Further Reading 277

Chapter 10 Gene Cloning in the Functional Analysis of Proteins

10.1 Introduction 279

10.2 Analyzing the Expression and Role of Unknown Genes 280

10.3 Determining the Cellular Location of Proteins 290

10.4 Mapping of Membrane Proteins 293

10.5 Detecting Interacting Proteins 297

10.6 Site-Directed Mutagenesis for Detailed Probing of Gene and Protein Function 304

Questions and Answers 309

Further Reading 312

Chapter 11 The Analysis of the Regulation of Gene Expression

11.1 Introduction 315

11.2 Determining the Transcription Start of a Gene 318

11.3 Determining the Level of Gene Expression 326

11.4 Identifying the Important Regulatory Regions 338

11.5 Identifying Protein Factors 350

11.6 Global Studies of Gene Expression 353

Questions and Answers 361

Further Reading 364

Chapter 12 The Production and Uses of Transgenic Organisms

12.1 What is a Transgenic Organism? 365

12.2 Why Make Transgenic Organisms? 367

12.3 How are Transgenic Organisms Made? 377

12.4 Drawbacks and Problems 396

12.5 Knockout Mice and Other Organisms: The Growth of Precision in Transgene Targeting 398

12.6 Is the Technology Available to Produce Transgenic People? 406

Questions and Answers 407

Further Reading 410

Chapter 13 Forensic and Medical Applications

13.1 Introduction 411

13.2 Forensics 411

13.3 DNA Profiling 413

13.4 Multiplex PCR 414

13.5 Samples for Forensic Analysis 415

13.6 Obtaining More Information from DNA Profiles 416

13.7 Other Applications of DNA Profiling 417

13.8 Medical Applications 418

13.9 Techniques for Diagnosis of Inherited Disorders 422

13.10 Whole Genome Amplification 435

13.11 Diagnosis of Infectious Disease 437

13.12 Diagnosis and Management of Cancer 439

Questions and Answers 441

Further Reading 444

Glossary 445

Index 453