An Introduction to Genetic Engineering

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ISTUDY
ISTUDY
 An Introduction to Genetic Engineering
Fourth Edition
The fourth edition of this popular textbook retains its focus on the
fundamental principles of gene manipulation, providing an accessible
and broad-based introduction to the subject for beginning undergraduate
students. It has been brought thoroughly up to date with new chapters on
the story of DNA and genome editing, and new sections on bioethics,
significant developments in sequencing technology and structural, func-
tional and comparative genomics and proteomics, and the impact of
transgenic plants. In addition to chapter summaries, learning objectives,
concept maps, glossary and key word lists, the book now also features new
concluding sections, further reading lists and websearch activities for
each chapter to provide a comprehensive suite of learning resources to
help students develop a flexible and critical approach to the study of
genetic engineering.

Desmond S. T. Nicholl was Senior Lecturer in Biological Sciences, Head of

Bioscience, Head of Quality Enhancement and Assistant Dean for
Education at the University of the West of Scotland. As well as three
previous editions of An Introduction to Genetic Engineering, he also authored
Cell and Molecular Biology (Learning & Teaching Scotland, 2000).

ISTUDY
 ‘Genetic engineering represents a toolbox that all students within the
basic and applied biology fields must get acquainted with. The fourth
edition of An Introduction to Genetic Engineering is an excellent up-to-date
version of a classic textbook. This ambitious book excellently balances
the molecular biology knowledge required to grasp the comprehensive
gene technology toolbox with a discussion of its impact on society.’
Per Amstrup Pedersen, University of Copenhagen

‘As a biomedical engineering professor teaching an undergraduate

Genetic Engineering course for close to 10 years, I use Dr Nicholl’s An
Introduction to Genetic Engineering as my go-to textbook. It is not one of
those overly thick textbooks that overwhelm students. Its
comprehensiveness captures readers’ attention with succinct
fundamental concepts that truly promote one’s interest in exploring
the wonder of many genetic engineering techniques and applications.
To facilitate that further, the material provided at the end of each
chapter encourages readers to expand their learning with relevant
resources ... Many of my students become so interested that they
pursue graduate degrees and have a career in this field. Dr Nicholl’s
textbook has a long-term influence on its readers.’
M. Ete Chan, State University of New York at Stony Brook

‘Dr Nicholl’s book covers all the basic material that one would expect
from its title, but what particularly impressed me was how it isn’t afraid
to move into political and socio-economic arenas. In Chapter 16, for
example, balanced arguments are presented for and against the
development of transgenic organisms, and these don’t always come out
in favour of the science.’
Neil Crickmore, University of Sussex

ISTUDY
 An Introduction to
Genetic Engineering
Fourth Edition

Desmond S. T. Nicholl

ISTUDY
 Shaftesbury Road, Cambridge CB2 8EA, United Kingdom
One Liberty Plaza, 20th Floor, New York, NY 10006, USA
477 Williamstown Road, Port Melbourne, VIC 3207, Australia
314–321, 3rd Floor, Plot 3, Splendor Forum, Jasola District Centre,
New Delhi – 110025, India
103 Penang Road, #05–06/07, Visioncrest Commercial, Singapore 238467

Cambridge University Press is part of Cambridge University Press & Assessment,

a department of the University of Cambridge.
We share the University’s mission to contribute to society through the pursuit of
education, learning and research at the highest international levels of excellence.

www.cambridge.org
Information on this title: www.cambridge.org/highereducation/isbn/9781009180597
DOI: 10.1017/9781009180610
First and second editions © Cambridge University Press 1994, 2002
Third and fourth editions © Desmond S. T. Nicholl 2008, 2023
This publication is in copyright. Subject to statutory exception and to the provisions
of relevant collective licensing agreements, no reproduction of any part may take
place without the written permission of Cambridge University Press & Assessment.
First published 1994
Second edition 2002
Third edition 2008
Fourth edition 2023
Printed in the United Kingdom by TJ Books Limited, Padstow, Cornwall, 2023
A catalogue record for this publication is available from the British Library.
A Cataloging-in-Publication data record for this book is available from the Library of Congress
ISBN 978-1-009-18059-7 Hardback
ISBN 978-1-009-18060-3 Paperback
Additional resources for this publication at www.cambridge.org/nicholl4
Cambridge University Press & Assessment has no responsibility for the persistence
or accuracy of URLs for external or third-party internet websites referred to in this
publication and does not guarantee that any content on such websites is, or will remain,
accurate or appropriate.

ISTUDY
 Contents

Preface page xv

Part 1 Genetic Engineering in Context

Chapter 1 Introduction 2

Chapter 2 The Story of DNA 16

Chapter 3 Brave New World or Genetic Nightmare? 36

Part 2 The Basis of Genetic Engineering

Chapter 4 Introducing Molecular Biology 52

Chapter 5 The Tools of the Trade 78

Chapter 6 Working with Nucleic Acids 94

Part 3 The Methodology of Gene Manipulation

Chapter 7 Host Cells and Vectors 134

Chapter 8 Cloning Strategies 160

Chapter 9 The Polymerase Chain Reaction 188

Chapter 10 Selection, Screening and Analysis

of Recombinants 210

Chapter 11 Bioinformatics 230

Chapter 12 Genome Editing 248

Part 4 Genetic Engineering in Action

Chapter 13 Investigating Genes, Genomes

and ‘Otheromes’ 264

Chapter 14 Genetic Engineering and Biotechnology 296

Chapter 15 Medical and Forensic Applications of

Gene Manipulation 326

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 vi CONTENTS

Chapter 16 Transgenic Plants and Animals 362

Chapter 17 The Other Sort of Cloning 390

Glossary 405
Index 439

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 Detailed Contents

Preface page xv

Part 1 Genetic Engineering in Context

Chapter 1 Introduction 2

Chapter Summary 2
1.1 What Is Genetic Engineering? 3
1.2 Laying the Foundations 5
1.3 First Steps in DNA Cloning 6
1.4 Using the Web to Support Your Studies 8
1.5 Conclusion: The Breadth and Scope of Genetic Engineering 12
Further Reading 13
Websearch 14
Concept Map 15

Chapter 2 The Story of DNA 16

Chapter Summary 16
2.1 How Science Works 17
2.1.1 A Simple Model for the Scientific Method 23
2.1.2 A More Realistic Model for How Science Works 24
2.2 DNA: A Biographical Timeline 25
2.3 People, Places and Progress: Paradigm Shifts or Step-
Changes? 28
2.4 Conclusion: The Scientific Landscape 32
Further Reading 33
Websearch 34
Concept Map 35

Chapter 3 Brave New World or Genetic Nightmare? 36

Chapter Summary 36
3.1 What Is Ethics? 37
3.1.1 The Ethical Framework 38
3.1.2 Is Science Ethically and Morally Neutral? 39
3.1.3 The Scope of Bioethics 40
3.2 Elements of the Ethics Debate 42
3.2.1 The Role of the Scientist 42
3.2.2 The Role of Society 43
3.2.3 Current Issues in Bioethics 43
3.3 Conclusion: Has Frankenstein’s Monster Escaped from
Pandora’s Box? 46
Further Reading 47
Websearch 47
Concept Map 49

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 viii DETAILED CONTENTS

Part 2 The Basis of Genetic Engineering

Chapter 4 Introducing Molecular Biology 52

Chapter Summary 52
4.1 How Living Systems Are Organised 53
4.2 The Flow of Genetic Information 55
4.3 The Structure of DNA and RNA 57
4.4 Gene Organisation 60
4.4.1 The Anatomy of a Gene 61
4.4.2 Gene Structure in Prokaryotes 62
4.4.3 Gene Structure in Eukaryotes 63
4.5 Gene Expression 64
4.5.1 From Genes to Proteins 65
4.5.2 Transcription and Translation 66
4.5.3 Regulation of Gene Expression 67
4.6 Genes and Genomes 69
4.6.1 Genome Size and Complexity 70
4.6.2 Genome Organisation 71
4.6.3 The Transcriptome and Proteome 72
4.7 Conclusion: Structure and Function 73
Further Reading 74
Websearch 75
Concept Map 76

Chapter 5 The Tools of the Trade 78

Chapter Summary 78
5.1 Restriction Enzymes – Cutting DNA 79
5.1.1 Type II Restriction Endonucleases 80
5.1.2 Use of Restriction Endonucleases 81
5.1.3 Restriction Mapping 84
5.2 DNA Modifying Enzymes 84
5.2.1 Nucleases 85
5.2.2 Polymerases 86
5.2.3 Enzymes That Modify the Ends of DNA Molecules 87
5.3 DNA Ligase – Joining DNA Molecules 88
5.4 Conclusion: The Genetic Engineer’s Toolkit 88
Further Reading 90
Websearch 91
Concept Map 92

Chapter 6 Working with Nucleic Acids 94

Chapter Summary 94
6.1 Evolution of the Laboratory 95
6.2 Isolation of DNA and RNA 99
6.3 Handling and Quantification of Nucleic Acids 100

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 DETAILED CONTENTS ix

6.4 Labelling Nucleic Acids 101

6.4.1 Types of Label – Radioactive or Not? 102
6.4.2 End Labelling 103
6.4.3 Nick Translation 104
6.4.4 Labelling by Primer Extension 104
6.5 Nucleic Acid Hybridisation 106
6.6 Gel Electrophoresis 108
6.7 DNA Sequencing: The First Generation 110
6.7.1 Principles of First-Generation DNA Sequencing 111
6.7.2 Sanger (Dideoxy or Enzymatic) Sequencing 112
6.7.3 Electrophoresis and Reading of Sequences 112
6.7.4 Automation and Scale-Up of DNA Sequencing 114
6.8 Next-Generation Sequencing Technologies 115
6.8.1 NGS – A Step-Change in DNA Sequencing 116
6.8.2 Principles of NGS 116
6.8.3 NGS Methodologies 119
6.9 Conclusion: Essential Techniques and Methods 127
Further Reading 129
Websearch 129
Concept Map 131

Part 3 The Methodology of Gene Manipulation

Chapter 7 Host Cells and Vectors 134

Chapter Summary 134

7.1 Types of Host Cell 135
7.1.1 Prokaryotic Hosts 136
7.1.2 Eukaryotic Hosts 136
7.2 Plasmid Vectors for Use in E. coli 137
7.2.1 What Are Plasmids? 137
7.2.2 Basic Cloning Plasmids 138
7.2.3 Slightly More Exotic Plasmid Vectors 139
7.3 Bacteriophage Vectors for Use in E. coli 141
7.3.1 What Are Bacteriophages? 141
7.3.2 Vectors Based on Bacteriophage λ 145
7.3.3 Vectors Based on Bacteriophage M13 147
7.4 Other Vectors 148
7.4.1 Hybrid Plasmid/Phage Vectors 148
7.4.2 Vectors for Use in Eukaryotic Cells 149
7.4.3 Artificial Chromosomes 150
7.5 Getting DNA into Cells 152
7.5.1 Transformation and Transfection 152
7.5.2 Packaging Phage DNA In Vitro 153
7.5.3 Alternative DNA Delivery Methods 154
7.6 Conclusion: From In Vitro to In Vivo 156
Further Reading 157
Websearch 157
Concept Map 159

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 x DETAILED CONTENTS

Chapter 8 Cloning Strategies 160

Chapter Summary 160

8.1 Which Approach Is Best? 161
8.1.1 Cloning in the Pre-genomic Era 162
8.1.2 Cloning (or Not) in the Genomic and Post-genomic Eras 162
8.2 Generating DNA Fragments for Cloning 165
8.2.1 Genomic DNA 165
8.2.2 Synthesis of cDNA 165
8.2.3 PCR Fragments 168
8.2.4 Synthetic Biology: Making Genes from Scratch 168
8.3 Inserting DNA fragments into Vectors 169
8.3.1 Ligation of Blunt/Cohesive-Ended Fragments 169
8.3.2 Homopolymer Tailing 170
8.3.3 Linkers and Adapters 170
8.3.4 Other Methods for Joining DNA Fragments and Vectors 173
8.4 Putting It All Together 175
8.4.1 Cloning in a λ Replacement Vector 176
8.4.2 Expression of Cloned cDNA Molecules 177
8.4.3 Cloning Large DNA Fragments in BAC and YAC Vectors 178
8.4.4 Gateway Cloning Technology 180
8.4.5 Golden Gate Cloning and Assembly 180
8.4.6 The Gibson Assembly Method 183
8.5 Conclusion: Designing a Cloning Strategy 184
Further Reading 184
Websearch 185
Concept Map 186

Chapter 9 The Polymerase Chain Reaction 188

Chapter Summary 188

9.1 History of the PCR 189
9.2 The Methodology of the PCR 190
9.2.1 Essential Features of the PCR 190
9.2.2 Designing Primers for the PCR 192
9.2.3 DNA Polymerases for the PCR 194
9.3 More Exotic PCR Techniques 195
9.3.1 PCR Using mRNA Templates 195
9.3.2 Nested PCR 198
9.3.3 Inverse PCR 199
9.3.4 Quantitative and Digital PCR 199
9.3.5 RAPD and Several Other Acronyms 202
9.4 Processing and Analysing PCR Products 205
9.5 Conclusion: The Game-Changing Impact of the PCR 205
Further Reading 206
Websearch 207
Concept Map 208

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 DETAILED CONTENTS xi

Chapter 10 Selection, Screening and Analysis

of Recombinants 210

Chapter Summary 210

10.1 Genetic Selection and Screening Methods 212
10.1.1 Use of Chromogenic Substrates 212
10.1.2 Insertional Inactivation 213
10.1.3 Complementation of Defined Mutations 214
10.1.4 Other Genetic Selection Methods 215
10.2 Screening Using Nucleic Acid Hybridisation 216
10.2.1 Nucleic Acid Probes 216
10.2.2 Screening Clone Banks 218
10.3 Use of the PCR in Screening Protocols 220
10.4 Immunological Screening for Expressed Genes 221
10.5 Analysis of Cloned Genes 222
10.5.1 Restriction Mapping 222
10.5.2 Blotting Techniques 223
10.5.3 Sub-cloning 225
10.5.4 DNA Sequencing 225
10.6 Conclusion: Needles in Haystacks 226
Further Reading 227
Websearch 227
Concept Map 229

Chapter 11 Bioinformatics 230

Chapter Summary 230

11.1 What Is Bioinformatics? 231
11.1.1 Computing Technology 232
11.1.2 The Impact of the Internet and World Wide Web 234
11.2 Biological Data Sets 234
11.2.1 Generation and Organisation of Information 234
11.2.2 Primary and Secondary Databases 235
11.2.3 Nucleic Acid Databases 236
11.2.4 Protein Databases 237
11.2.5 Other Bioinformatics Resources 239
11.3 Using Bioinformatics as a Tool 241
11.3.1 Avoiding the ‘GIGO’ Effect – Real Experiments 241
11.3.2 Avoiding the Test Tube – Computational Experimentation 242
11.3.3 Presentation of Database Information 243
11.4 Conclusion: Bioscience and ‘Big Data’ 244
Further Reading 245
Websearch 246
Concept Map 247

Chapter 12 Genome Editing 248

Chapter Summary 248

12.1 Gene Targeting 250
12.2 Genome Editing Using Engineered Nucleases 251
12.2.1 Zinc-Finger Nucleases

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 xii DETAILED CONTENTS

12.2.2 TALENs 253

12.2.3 The CRISPR-Cas9 System 253
12.2.4 Prime Editing 256
12.3 Editing RNA as an Option 258
12.4 Where Can Genome Editing Take Us? 258
12.5 Conclusion: From Genome Read to Genome Write 259
Further Reading 260
Websearch 260
Concept Map 261

Part 4 Genetic Engineering in Action

Chapter 13 Investigating Genes, Genomes

and ‘Otheromes’ 264

Chapter Summary 264

13.1 Analysis of Gene Structure and Function 265
13.1.1 A Closer Look at Sequences 265
13.1.2 Finding Important Regions of Genes 266
13.1.3 Investigating Gene Expression 270
13.2 Understanding Genomes 272
13.2.1 Analysing and Mapping Genomes 273
13.2.2 An Audacious Idea 276
13.2.3 The Human Genome Project 277
13.2.4 Other Genome Projects 281
13.3 ‘Otheromes’ 282
13.3.1 The Transcriptome 282
13.3.2 The Proteome 285
13.3.3 Metabolomes, Interactomes and More 286
13.4 Life in the Post-genomic Era 288
13.4.1 Structural Genomics and Proteomics 289
13.4.2 Functional Genomics 289
13.4.3 Comparative Genomics 289
13.5 Conclusion: The Central Role of the Genome 291
Further Reading 292
Websearch 292
Concept Map 294

Chapter 14 Genetic Engineering and Biotechnology 296

Chapter Summary 296

14.1 Making Proteins 297
14.1.1 Native and Fusion Proteins 299
14.1.2 Yeast Expression Systems 300
14.1.3 The Baculovirus Expression System 301
14.1.4 Mammalian Cell Lines 302
14.2 Protein Engineering 303
14.2.1 Rational Design 303
14.2.2 Directed Evolution 305

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 DETAILED CONTENTS xiii

14.3 From Laboratory to Production Plant 308

14.3.1 Thinking Big – The Biotechnology Industry 308
14.3.2 Production Systems 310
14.3.3 Scale-Up Considerations 310
14.3.4 Downstream Processing 312
14.4 Examples of Biotechnological Applications of
rDNA Technology 312
14.4.1 Production of Enzymes 313
14.4.2 The BST Story 314
14.4.3 Therapeutic Products for Use in Human Healthcare 316
14.4.4 Meeting the COVID-19 Challenge 320
14.5 Conclusion: Industrial-Scale Biology 322
Further Reading 323
Websearch 324
Concept Map 325

Chapter 15 Medical and Forensic Applications

of Gene Manipulation 326

Chapter Summary 326

15.1 Diagnosis and Treatment of Medical Conditions 327
15.1.1 Diagnosis of Infection 327
15.1.2 Patterns of Inheritance 328
15.1.3 Genetically Based Disease Conditions 330
15.1.4 Investigating Disease Alleles Using Comparative Genomics 337
15.1.5 Vaccine Development Using rDNA 338
15.1.6 Therapeutic Antibodies 339
15.1.7 Xenotransplantation 341
15.2 Treatment Using rDNA Technology – Gene Therapy 342
15.2.1 Getting Transgenes into Patients 343
15.2.2 Gene Therapy for Adenosine Deaminase Deficiency 344
15.2.3 Gene Therapy for Cystic Fibrosis 346
15.2.4 What Does the Future Hold for Gene Therapy? 346
15.3 RNA Interference 347
15.3.1 What Is RNAi? 347
15.3.2 Using RNAi as a Tool for Studying Gene Expression 348
15.3.3 RNAi as a Potential Therapy 348
15.3.4 Antisense Oligonucleotides 350
15.4 Medical Applications of Genome Editing 350
15.4.1 Disease Targets for Genome Editing 350
15.4.2 Sickle-Cell Success 351
15.4.3 CRISPR-Cas9 – CAR T-Cell Therapies in Cancer Treatment 352
15.4.4 The CCR5 Controversy 353
15.5 DNA Profiling 354
15.5.1 The History of ‘Genetic Fingerprinting’ 354
15.5.2 DNA Profiling and the Law 356
15.5.3 Mysteries of the Past Revealed by Genetic Detectives 356
15.6 Conclusion: rDNA in Diagnosis, Analysis and Treatment 358
Further Reading 359
Websearch 360
Concept Map

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 xiv DETAILED CONTENTS

Chapter 16 Transgenic Plants and Animals 362

Chapter Summary 362

16.1 A Complex Landscape 363
16.2 Transgenic Plants 365
16.2.1 Why Transgenic Plants? 365
16.2.2 Making Transgenic Plants 365
16.2.3 Putting the Technology to Work 369
16.2.4 Have Transgenic Plants Delivered or Disappointed? 377
16.3 Transgenic Animals 378
16.3.1 Why Transgenic Animals? 378
16.3.2 Producing Transgenic Animals 379
16.3.3 Applications of Transgenic Animal Technology 380
16.4 Future Trends 383
16.4.1 Transgenesis or Genome Editing? 384
16.4.2 Gene Drives 384
16.5 Conclusion: Changing Genomes and Attitudes 385
Further Reading 386
Websearch 387
Concept Map 389

Chapter 17 The Other Sort of Cloning 390

Chapter Summary 390

17.1 Early Thoughts and Experiments 391
17.1.1 First Steps towards Cloning 393
17.1.2 Nuclear Totipotency 393
17.2 Frogs and Toads and Carrots 394
17.3 A Famous Sheep – The Breakthrough Achieved 396
17.4 Beyond Dolly 398
17.4.1 Potential Unfulfilled? 399
17.4.2 The Future of Organismal Cloning 400
17.5 Conclusion: From Genome to Organism 401
Further Reading 402
Websearch 403
Concept Map 404

Glossary 405
Index 439

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 Preface

Advances in genetics continue to be made at an ever increasing rate,

which presents something of a dilemma when writing an introduc-
tory text on the subject. In the years since the third edition was
published, many new applications of gene manipulation technology
have been developed; genome sequencing has become available at
bench-top scale and cost, and gene editing can be achieved using very
modest laboratory infrastructure. Personal genome profiling is avail-
able from a range of companies, and genetic technology has played a
major role in managing many aspects of the COVID-19 pandemic,
from diagnostic testing to rapid development of safe and effective
vaccines.
Information technology resources, coupled with the internet and
World Wide Web, have been critical parts of all these developments,
providing tools for the analysis of DNA sequences and instant sharing
of data across the globe. At the same time, a level of mistrust has
developed among some sections of society, largely driven by misinfor-
mation on social media channels, which has illustrated the power of
the internet in a less positive way. It is against this background that
some themes began to emerge for the fourth edition, reflecting the
aim of encouraging students to use the excellent resources on the
web, whilst retaining a level of critical assessment of the information.
Aspects around the ethics of using genetic technology are perhaps
now even more important than before, so these are discussed early in
the text to enable the applications to be placed within an appreciation
of the ethical framework.
Whilst aiming for a slight broadening in scope, I remain convinced
that a basic technical introduction to the subject should be the major
focus of the text. Thus, some of the original methods used in gene
manipulation have been kept as examples of how the technology
developed, even though some of these have become little used or even
obsolete. From the educational point of view, this should help the
reader cope with more advanced information about the subject, as a
sound grasp of the basic principles is an important part of any intro-
duction to genetic engineering. I have been gratified by the many
positive comments about the third edition of the text, and I hope that
this new edition continues to serve a useful purpose as part of the
introductory literature on this fascinating subject.
This book is organised as four parts. Part 1 (Genetic Engineering in
Context; Chapters 1–3) sets the scene and brings the discussion of the
ethical issues around DNA technology to the start of the book. Part 2
(The Basis of Genetic Engineering; Chapters 4–6) provides an introduction
to molecular biology and outlines the tools available to the genetic
engineer, and Part 3 (The Methodology of Gene Manipulation; Chapters
7–12) extends this theme further by examining how these tools enable

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 xvi PREFACE

sophisticated experiments and procedures to be carried out. Finally, in

Part 4 (Genetic Engineering in Action; Chapters 13–17), we look at the
impact of DNA technology across a range of key areas.
In the fourth edition, I have expanded the range of features that
should be useful as study aids where the text is used to support a
particular academic course. In the book, there are text boxes sprinkled
throughout the chapters. These highlight key points on the way
through the text, and can be used as a means of summarising the
content. At the start of each chapter, the aims of the chapter are
presented, along with a chapter summary in the form of learning object-
ives. These have been written quite generally, so that an instructor can
modify them to suit the level of detail required. A list of the key words
in each chapter is also provided for reference. These are shown as bold
in the text; terms in blue can also be found in the Glossary. A new
addition to the end of each chapter is a websearch page that provides
some structured web-based search exercises that help to set the chap-
ter in context and act as a start point for further study using the
resources available online. As in previous editions, a concept map has
been generated for each chapter, showing how the main topics are
linked. The concept maps provided here are essentially summaries of
the chapters, and may be examined either before or after reading
the chapter.
As this remains an introductory text, no in-text reference has been
made to the primary (research) literature, but some suggestions for
further reading are given at the end of each chapter. Most of these are
available in open-access format or may be available through an insti-
tution’s library subscription service. A glossary of terms used has also
been provided.
A new development for the fourth edition is a set of online resources
at www.cambridge.org/nicholl4. This provides access to a range of
materials from the book (and additional information) that I hope will
be useful in building a learning system to suit your preferred learning
style. The resources have been provided in electronic format as a study
guide to enable collation into a set of student-generated notes.
My thanks go to the anonymous (but appreciated) reviewers of the
proposal and the early versions of the manuscript. Their comments
and suggestions have made the book better; any errors of fact or
interpretation of course remain my own responsibility. Special thanks
to Megan Keirnan, Susan Francis, Helen Shannon and Rachel Norridge
at Cambridge University Press, and to Joyce Cheung, for their cheerful
advice, support, encouragement and patience, which helped bring the
project to its conclusion.
My final and biggest thank you goes as ever to my wife Linda and to
Charlotte, Thomas and Anna, who have grown up along with the
various editions of ‘IGE’. I dedicate this new edition to them.

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 Part 1
Genetic Engineering
in Context

Chapter 1 Introduction 2
Chapter 2 The Story of DNA 16
Chapter 3 Brave New World or Genetic Nightmare? 36

ISTUDY
 Chapter 1 Summary
Learning Objectives
When you have completed this chapter, you will be able to:
r Define genetic engineering as it will be described in this book
r Outline the basic features of genetic engineering
r Describe the emergence of gene manipulation technology
r Explain the steps required to clone a gene
r Appreciate elements of the ethical debate surrounding genetic
engineering
r Identify a range of internet-based resources related to DNA
technology

Key Words

Genetic engineering, bioinformatics, gene manipulation, gene

cloning, recombinant DNA (rDNA) technology, genetic
modification, new genetics, DNA technology, molecular
agriculture, genethics, Gregor Mendel, James Watson, Francis
Crick, DNA ligase, type II restriction enzyme, plasmid,
extrachromosomal element, replicon, clone, genetically modified
organism (GMO), internet, World Wide Web, Tim Berners-Lee,
uniform resource locator (URL), domain (re. URL), search engine,
valid, reliable, peer review, Encyclopedia Britannica, Wikipedia,
social media, misinformation, disinformation, suggested search
term (SST), digital object identifier (DOI).

ISTUDY
 Chapter 1

Introduction
1.1 What Is Genetic Engineering?

Making progress in any scientific discipline depends on continually

developing techniques and methods to extend the range and sophisti-
cation of experiments that may be performed. Within the biosciences,
this has been demonstrated in a spectacular way by the emergence
and development of genetic engineering. In 2022, we marked the
fiftieth anniversary of the creation of the first recombinant DNA
molecules, an event that is often used to note the start of the recom-
binant DNA era of genetics. The five decades since 1972 have seen
astonishing progress in the breadth and scope of the technology, and
it is now routine practice to identify a specific DNA fragment from the
genome of an organism, determine its base sequence and assess its
function. The sequence might then be altered and replaced into the
organism it came from, or a different organism, to achieve a particu-
lar goal. We have seen the expansion of the technology into the
domain of ‘big science’ in the era of the Human Genome Project,
and its return to the small-scale laboratory as new developments have
appeared. Whole genomes can now be sequenced using a benchtop
machine, and genome editing enables researchers to alter the genome
of an organism with a high level of precision. All of this is now
underpinned by the astonishing developments in bioinformatics,
with sophisticated computational tools available to analyse almost
unimaginable amounts of data that are generated on a daily basis.
The term genetic engineering is often thought to be rather emotive Several terms may be used to
or even trivial, yet it is probably the label that most people would describe the technologies
recognise. However, there are several other terms that can be used to involved in manipulating genes.
describe the technology, including gene manipulation, gene cloning,
recombinant DNA (rDNA) technology and genetic modification. You
may also come across the term the ‘new genetics’, although we are at
a point where this is perhaps less useful than was the case previously.
A more useful generic term that covers a wide range of techniques and
applications is simply DNA technology. There are also legal defin-
itions used in administering regulatory mechanisms in countries
where genetic engineering is practised.

ISTUDY
 4 1 INTRODUCTION
The genetic material provides a
rich resource in the form of
information encoded by the
sequence of bases in the DNA.
Gene cloning enables isolation
and identification of
individual genes.
Fig. 1.1 The four steps in cloning
a DNA sequence. Steps 1 and 2 are
carried out in vitro and generate the
recombinant DNA molecules.
A host organism, such as a
bacterium, is used for steps 3 and 4
(in vivo). The term clone refers to
the colonies of identical host cells
produced during amplification of
the cloned fragments. The cloned
sequence can then be isolated and
processed further. Gene cloning is
sometimes referred to as molecular
cloning, to distinguish the process
from the cloning of whole
organisms.
ISTUDY
Although there are many diverse and complex techniques
involved, the basic principles of genetic manipulation are reasonably
simple. The premise on which the technology is based is that genetic
information, encoded by DNA and arranged in the form of genes, is a
resource that can be manipulated in various ways to achieve certain
goals in both pure and applied science, medicine, biotechnology and
agriculture. There are many areas in which genetic manipulation has
made a significant impact, including:
r Basic research on gene structure and function
r Production of useful proteins by novel methods
r Generation of transgenic plants and animals
r Medical diagnosis and treatment
r Forensic analysis of crime scene samples
r Molecular anthropology and the study of evolution
r Genome analysis and genome editing
In later chapters, we will look at how DNA technology has contributed
to these areas.
The mainstay of genetic manipulation is the ability to isolate a
single DNA sequence from the genome. This is the essence of gene
cloning and can be considered as a series of four steps (Fig. 1.1).
Successful completion of these steps provides the genetic engineer
with a specific DNA sequence, which may then be used for a variety
of purposes. A useful analogy is to consider gene cloning as a form of
molecular agriculture, enabling the production of large amounts (in
genetic engineering, this means nanograms or micrograms) of a par-
ticular DNA sequence. Although the basic cloning methodology has
been extended (and in many cases replaced) by technologies such as
the polymerase chain reaction, large-scale DNA sequencing and
genome editing, this ability to isolate a particular gene sequence is
Source of DNA
1 Generation of DNA fragments
In vitro
(test tube)
2 Joining to vector
3 Introduction into host cell
In vivo
(organism)
4 Selection of cloned sequence
Further processing
 1.2 LAYING THE FOUNDATIONS 5

still a major part of gene manipulation as carried out on a day-to-day

basis in research laboratories worldwide.
One aspect of genetic engineering that has given cause for concern
As well as technical and scientific
is the debate surrounding the potential applications of the technology. challenges, modern genetics
The term genethics is sometimes used to describe the ethical prob- poses many moral and ethical
lems that exist in modern genetics, which are likely to increase in questions.
both number and complexity as genetic engineering technology
becomes more sophisticated and implemented more widely. The use
of transgenic plants and animals, investigation of the human genome,
gene therapy, genome editing and many other topics are of concern
not just to the scientist, but also to the population as a whole.
Developments in genetically modified foods have provoked a well-
documented public backlash against the technology in many coun-
tries. Additional developments in the cloning of organisms, and in
areas such as in vitro fertilisation and xenotransplantation, raise fur-
ther questions. Although not strictly part of gene manipulation tech-
nology, organismal cloning will be considered later in this book, as
this is an area of much concern and can be considered as genetic
engineering in its broadest sense. Research on embryonic stem cells,
and the potential therapeutic benefits that this may bring, is another
area of concern that is part of the general advance of genetics. We will
look at some of these ethical aspects in more detail in Chapter 3.

1.2 Laying the Foundations

Although the techniques used in gene manipulation began to appear

Gregor Mendel is often
in the 1970s, we should remember that development of these tech- considered the ‘father’
niques depended on the knowledge and expertise provided by chem- of genetics.
ists, biochemists and microbial geneticists working in the earlier
decades of the twentieth century. We can consider the development
of genetics as falling into three main eras (Fig. 1.2). The science of
genetics really began with the rediscovery of Gregor Mendel’s work at
the start of the century, and the next 40 years or so saw the elucida-
tion of the principles of inheritance and genetic mapping. Microbial
genetics became established in the mid-1940s, and the role of DNA as
the genetic material was confirmed. During this period, great
advances were made in understanding the mechanisms of gene trans-
fer between bacteria, and a broad knowledge base was established,
from which later developments would emerge.
Determination of the structure of DNA by James Watson and Watson and Crick’s double helix
Francis Crick in 1953 provided the stimulus for the development of is perhaps the most ‘famous’ and
genetics at the molecular level, and the next few years saw a period of most easily recognised molecule
intense activity and excitement as the main features of the gene and in the world.
its expression were determined. This work culminated in the
deciphering of the complete genetic code in 1966, and the stage was
now set for the appearance of the new discoveries that would lead to
the development of the early techniques in recombinant DNA
technology.

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 6 1 INTRODUCTION
Fig. 1.2 The history of genetics
since 1900. Three eras can be
identified. Darker shaded areas
represent the periods of major
development in each branch of the
subject, although advances continue
to be made in all of these areas.
By the end of the 1960s, most of
the essential requirements for
the emergence of gene
technology were in place.
ISTUDY
Mendelian or classical genetics
1900
1910 Genetic mapping
1920
Transformation demonstrated
1930
1940 Microbial and molecular genetics
1950
Structure of the double helix
1960
Genetic code
1970 Gene manipulation
Gene cloning
1980 DNA sequencing
1990 Human Genome Project
Internet era
2000
2010 Genome editing
2020
1.3 First Steps in DNA Cloning
In the late-1960s, there was a sense of frustration among scientists
working in the field of molecular biology. Research had developed to
the point where progress was being hampered by technical con-
straints, as the elegant experiments that had helped to decipher the
genetic code could not be extended to investigate the gene in more
detail. However, a number of developments provided the necessary
stimulus for gene manipulation to become a reality. In 1967, the
enzyme DNA ligase was isolated. This enzyme can join two strands
of DNA together, a prerequisite for the construction of recombinant
molecules, and can be regarded as a sort of molecular glue. This was
followed by the isolation of the first type II restriction enzyme in
1970, a major milestone in the development of genetic engineering.
Restriction enzymes are essentially molecular scissors that cut DNA at
precisely defined sequences. Such enzymes can be used to produce
fragments of DNA that are suitable for joining to other fragments.
Thus, by 1970, the basic tools required for the construction of recom-
binant DNA were available.
The first recombinant DNA molecules were generated at Stanford
University in 1972, utilising the cleavage properties of restriction
enzymes (scissors) and the ability of DNA ligase to join DNA strands
together (glue). The importance of these first tentative experiments
cannot be overstated. Scientists could now join different DNA mol-
ecules together and could link the DNA of one organism to that of a
completely different organism. The methodology was extended in
 1.3 FIRST STEPS IN DNA CLONING 7

(a) Source DNA (b) Fig. 1.3 Cloning DNA fragments.

(a) The source DNA is isolated and
fragmented into suitably sized
pieces. This may be carried out
mechanically or by using restriction
enzymes. (b) The fragments are
then joined to a carrier molecule or
vector to produce recombinant
DNA molecules. In this case, a
plasmid vector is shown. (c) The
DNA fragments Join to vector
recombinant DNA molecules are
then introduced into a host cell
(a bacterial cell in this example) for
(c) (d) propagation as clones (d). These
can be stored as a stable resource
that can be used for further
analysis.

Introduce into host cell Grow clones

1973 by joining DNA fragments to the plasmid pSC101, which is an

The key to gene cloning is
extrachromosomal element derived from an antibiotic resistance ensuring that the target sequence
plasmid originally isolated from the bacterium Salmonella typhimurium. is replicated in a suitable
These recombinant molecules behaved as replicons, i.e. they could host cell.
replicate when introduced into Escherichia coli cells. Thus, by creating
recombinant molecules in vitro, and placing the construct in a bacter-
ial cell where it could replicate in vivo, specific fragments of DNA could
be isolated from bacterial colonies that formed clones (colonies
formed from a single cell, in which all cells are identical) when grown
on agar plates. This development marked the emergence of the tech-
nology that became known as gene cloning (Fig. 1.3).
The discoveries of 1972 and 1973 triggered what is perhaps the
biggest scientific revolution of all – the ‘new genetics’ era had arrived.
The use of the new technology spread very quickly, and a sense of
urgency and excitement prevailed. This was dampened somewhat by
the realisation that the new technology could give rise to potentially The development and use of
harmful organisms with undesirable characteristics. It is to the credit GMOs pose some difficult ethical
of the biological community that measures were adopted to regulate questions that may not arise in
other areas such as gene cloning.
the use of gene manipulation, and that progress in contentious areas
was restricted until more information became available about the
possible consequences of the inadvertent release of organisms con-
taining recombinant DNA. However, the development of genetically
modified organisms (GMOs), particularly crop plants, has reopened
the debate about the safety of these organisms and the consequences

ISTUDY
 8 1 INTRODUCTION
Fig. 1.4 Eras of genomics.
Technological development in
DNA science and computation in
the 1970s and 1980s led to the
project to sequence the human
genome, which largely defined the
genomic era. The knowledge gained
has continued to drive the
development of techniques and
applications in the post-
genomic era.
ISTUDY
Restriction enzymes discovered
1970
rDNA molecules generated
Pre-genomic era
Rapid DNA sequencing developed
1980
First-generation sequencing technology
First viral genome sequenced
1990 Human Genome Project started
First bacterial genome sequenced
Genomic era
Second-generation sequencing technology
2000
Human genome completed
Third-generation sequencing technology
2010
Genome editing
Post-genomic era
Systems and synthetic biology
2020 First trial of gene editing in vivo
T2T human genome sequence
of releasing GMOs into the environment. In addition, many of the
potential medical benefits of gene manipulation, genetics and cell
biology pose ethical questions that may not be easy to answer. We
will come across some of these issues later in this book.
DNA technology continued (and continues) to expand at pace, with
a number of key techniques developed in the late-1970s and early-
1980s that would enable a step-change in scale to be achieved, with
the ambitious project to sequence the human genome being com-
pleted in 2003. Further developments have changed DNA sequencing
significantly, to the extent that we can now usefully describe DNA
technology as itself falling into three eras (Fig. 1.4). As we explore the
topics in this book, it may be useful to keep this diagram in mind as a
‘roadmap’ to help us place the technology in context.
1.4 Using the Web to Support Your Studies
Since the first edition of this book was published in 1994, the growth
and development of DNA technology has been impressive, and often
astonishing. In many ways, the parallel development of the internet
and World Wide Web (www) is equally impressive, and the gener-
ation of students who may be using this book has grown up in a world
that is completely immersed in the technology associated with ‘the
web’. Although information on how to access the internet is no longer
needed, some of the guidance for using the web that was included in
earlier editions remains appropriate.
The internet was conceived and developed by Tim Berners-Lee
(now Sir Tim) in 1989 whilst working at CERN in Geneva. If you are
not familiar with CERN and Berners-Lee, look these up now using your
computer, laptop, tablet or smartphone! The term internet is used to
 1.4 USING THE WEB TO SUPPORT YOUR STUDIES 9

describe the network of computers that together provide the means to

publish and share information, whilst the World Wide Web is a more
general description of the information that is published using the
internet. However, the two terms are often used interchangeably,
and the phrases ‘surfing the net’, ‘surfing the web’ and ‘look it up
online’ have become part of modern-day language. We will use ‘the
web’ in this book when referring to the World Wide Web.
A really positive feature of the web is the range of material that
can be found with a few clicks of a mouse or swipes with a finger. This book can be used as a guide
The fluidity and dynamic nature of the web mean that a printed book to explore topics more broadly,
(like this one) cannot possibly compete; so we will not try to. A subtle and/or in more depth, using
the web.
shift in emphasis is therefore required – this book is a resource that
can help steer you through the confusion of the web, as well as
providing a structured look at the subject matter. I have therefore
assumed that you will be reading this text with pretty much instant
and continuous access to the internet, and that you will be able to
use the web to help gather, collate and interpret additional infor-
mation that you find.
Finding websites is generally straightforward if you know where
you are going. Each website has an ‘address’ known as a uniform
resource locator (URL). A URL generally begins with http://www.
followed by the specific domain address. This may end with a country
identifier such as .uk, .ca, .cn, .us, .au, etc. The most common domain
ending is .com (where the term ‘dot com’ comes from), which is used
by around 50 per cent of all websites. Many student textbooks have
associated websites, as do research groups, university departments,
companies, etc.
If you don’t know the URL, one of the many search engines
enables you to look for information using a range of terms, and it is
astonishing what you can find. However, caution is needed: there is an
awful lot of information on the web, and there is a lot of awful infor-
mation there as well! It is very easy to get sidetracked and end up
wasting a lot of time searching through sites that are of no value (but
may be interesting nonetheless). As I write this, I have just typed in
the search terms that I used to illustrate this section in the third
edition of this book. The number of ‘hits’ for each of the various
terms, in 2007 and 2021, is shown in Table 1.1.
A number of points can be made when we consider the data.
Firstly, the number of retrieved items is often far too large to be
useful, and thus more specific, defined or restricted search terms will
generally produce fewer results. However, even with something like
‘sheep cloning’ or ‘plasmid vector’, there is still too much information
to look through, so learning to use the search and filter facilities
provided by various search engines and websites is well worth the
effort. Secondly, it seems that there has been an orders-of-magnitude
increase in the number of hits generated by all of these terms. At first
glance, this may not seem implausible, given the increase in research
and development that will have occurred in these areas since 2007.
However, we do need to be a little cautious about inferring too much,

ISTUDY
 10 1 INTRODUCTION

Table 1.1 Comparison of the number of web search ‘hits’

obtained using some search terms in 2007 and 2021

Search term ‘Hits’ 2007 ‘Hits’ 2021 Increase

genetics 5 564 000 578 000 000 104 
cloning 2 244 000 378 000 000 168 
cystic fibrosis 966 400 61 400 000 64 
DNA cloning 499 800 33 200 000 66 
sheep cloning 127 700 5 380 000 42 
plasmid vector 71 200 18 600 000 261 
homopolymer tailing 123 65 000 528 

Care is needed when
interpreting information from
the web. A critical approach is
always helpful.
for reasons that are not immediately obvious from the data presented.
Factors such as developments in web-searching techniques and the
algorithms used will have a major impact on the returns, and it is
unlikely that these variables were the same in 2007 and 2021. The
search engine used also has an impact – for a particular search term,
different variants return different numbers of hits, and even the same
search engine can return different numbers of hits for the same term
on different days. Thus, we can’t be sure that the information is an
accurate reflection of the actual number of items on a topic at any
point in time. Whilst it does give some indication of the expansion of
the science itself, its reporting across the web and its ‘searchability’, it
is of limited value. In fact, the comparison presented here is neither
valid nor reliable. These terms have specific meaning in scientific
methodology, which we will explore further in Section 2.1.
The main point to be aware of when using the web is there is very
little restriction on what gets published, and by whom. The peer review
system that is used in science is not often applied to web-based infor-
mation, so not all information may be accurate. The type of website
can often give some indication as to how reliable the information may
be. The last part of the URL usually indicates the sort of organisation,
although this does vary from country to country. Examples are .gov for
government sites, .ac.uk for UK academic sites (.edu used in some other
countries) and .com or .co for commercial or company sites. The char-
acteristics of these types of site can be described as follows:
r Government sites – usually accurate information, but this can have a
particular emphasis. Political influences and ‘spin’ can sometimes
make certain types of information highly suspect for some
government-controlled sites. A knowledge of the national political,
social and cultural situation may be useful in assessing the reliability
of government-controlled websites.
r Research institutes and universities – very often the most useful sites for
supporting academic study, as these offer a high level of specificity
and detail. They tend to be managed by people who are familiar with
publishing information via the peer review mechanism, and there-
fore are aware of the need to be accurate and unbiased.

ISTUDY
 1.4 USING THE WEB TO SUPPORT YOUR STUDIES 11

r Special interest groups/professional societies – similar to research insti-

tutes and universities in terms of authoritative and accurate infor-
mation. Can range from fairly broad to very narrowly
specific coverage.
r Company sites – again can provide a wealth of information, but this is
obviously presented to support company aims, ethos and policy in
most cases. Many responsible companies take care to present a
balanced view of any contentious issues, often including links to
other sources of information.
r Publishers – range from book publishers to online journals. Often
provide excellent resource material, but for some journal sites,
access may require registration and payment of a fee for each issue
or article downloaded.
r Information ‘gateway’ sites – many specialist research areas have asso-
ciated sites that collate and distribute information about the latest
developments. Often it is possible to register for email updates to be
sent directly when they are published. As with journals, some of
these sites require registration and subscription to access the full
range of facilities. May be associated with a special interest group or
professional body.
r Pressure group sites – can provide a lot of useful information, very
often gathered to counteract some particular claim made by another
party. Despite the obvious dangers of accepting the views presented,
many of these sites do take care to try to present accurate infor-
mation. Often there is a higher risk of confirmation bias (see Section
2.1) affecting content and opinion than in other types of website.
Views can range from moderate to extreme.
r Personal sites – prepared by individuals, therefore likely to be the
most variable in terms of content and viewpoint. Range from highly
respected sites by well-known experts to the sort of site that is best
avoided as far as serious study is concerned.

The difficulty in weeding out the less useful (or downright weird) sites
is that anyone with a flair for design can produce a website that looks
impressive and authoritative. Conversely, some extremely useful and
respected sites can look a little dull if a good web designer has not
been involved. However, as long as you remember that critical evalu-
ation is essential, you should have few problems.
Online encyclopedias are very useful as starting points for
researching a topic. Long-established versions such as Encyclopedia
Britannica are now available online, with others set up specifically for
the web environment (e.g. encyclopedia.com). The interactive encyclo-
pedia Wikipedia, which was set up in 2001, is ‘editable’ by its con-
tributors, and thus represents a novel publishing phenomenon. The
theory is that it is effectively a self-regulating project that grows as
more articles are added and refined. In general, the articles are writ-
ten by authors who are expert in their fields and who take care to
ensure accuracy and objectivity. This is strengthened by the post-
publication ‘peer-amended’ editing process (as opposed to the

ISTUDY
 12 1 INTRODUCTION
Social media sites present
particular challenges around how
we receive and process
information. Scientific topics, and
scientists themselves, can be
affected (often in a negative way)
by what circulates on social
media platforms.
ISTUDY
peer-reviewed pre-publication approach of more traditional publish-
ing). Wikipedia is therefore a useful resource but (as with any source)
should be backed up by further validation of the content if serious
study is undertaken on a particular topic.
There are, of course, less positive aspects of the web, so care is
needed. In particular, the all-pervasive nature of social media, and the
impact on how we receive, filter and process information, is perhaps
one of our most critical challenges in dealing with online information.
Whilst social media sites provide some tremendous benefits, they are
not often reliable sources for authoritative and objective information.
The spread of misinformation, disinformation, fake news stories,
conspiracy theories and the like can have a significant destabilising
influence across a wide spectrum of society, with international pene-
tration achieved easily via the web. A recent example affecting the
public perception of scientific information was the anti-vaccination
reaction to the SARS-CoV-2 viral pandemic; the spread of mis- and
disinformation around the vaccines developed to reduce the risk of
serious illness from COVID-19 undoubtedly led to additional loss of
life that could have been prevented.
Despite the challenges, part of the fun of using the web is that it is
such a diverse and extensive medium, and it is constantly changing and
expanding. This can present a problem when including URLs in a printed
text, as website addresses may change over time as sites develop, appear
and disappear. The ‘site not found’ message can be frustrating, so where
URLs are included in the book, these tend to be long-established sites that
are unlikely to change. In practice, it is now unusual to copy a URL
manually into a web browser from a printed source, and often the best
way to find the URLs in this text is to type in a search term related to the
site and select the matching URL from the list that appears. Thus, in
many cases where websites are suggested, I have included a suggested
search term (SST) instead of a URL. If you are using this book as a module
or course text, your tutor will be able to direct you to any specific
websites that support your course. Finally, using a search engine is the
most effective way of getting up-to-date information about what is
available, and by finding a few relevant sites, you can quickly build up
a set of links that take you into other related sites. Good surfing!
1.5 Conclusion: The Breadth and Scope
of Genetic Engineering
In this introduction, we have looked at genetic engineering in the
context of the history of genetics from 1900. The rediscovery of
Mendel’s work stimulated the study of patterns of inheritance in
organisms such as the fruit fly. In parallel, microbiologists began to
consider how bacteria might be useful as model systems for studying
genetics, and in 1928, transformation of bacterial characteristics was
demonstrated in one of the classic experiments in genetics.
 FURTHER READING 13

Progress in genetics is often dependent on the technology avail-

able; both ‘hard’ technology (such as laboratory and computing equip-
ment) and ‘soft’ technology (biological supplies such as enzymes) are
important. The start of the molecular genetics era is often dated to
1953 when Watson and Crick determined that DNA was a double
helix, using data from a number of sources, including the ratios of
the DNA bases in various organisms and X-ray crystallography of DNA
samples. Over the next 20 years, a series of elegant experiments
showed how DNA functions as the genetic material.
In 1972, the availability of type II restriction enzymes and DNA ligase
enabled the first rDNA molecules to be generated, and we entered the
era of DNA technology. Gene cloning enabled the isolation of specific
DNA sequences, and the structure of the gene was studied at the
molecular level for the first time. DNA sequencing developed from
a laborious and difficult process to a relatively straightforward one
with the advent of rapid sequencing methods in 1977, and within a
few years, automation and ambition had brought the previously
unthinkable within reach – determining the sequence of the human
genome. This astonishing feat was completed in 2003, although we will
see in Chapters 6, 11, 12 and 13 that the term ‘completed’ is not really
accurate and genome sequencing continues to expand and have a
significant impact on a number of different disciplines.
In this chapter, we have touched on the ethical questions that DNA
technology raises; we will examine some of these in more detail in
Chapter 3, and also when we consider the applications of genetic
engineering in Part 4. This aspect is a fundamental part of the deploy-
ment of any technology, but is particularly important when we are
dealing with things that can have a direct impact on human health-
care, the diagnosis and treatment of disease, reproduction, nutrition
and forensic analysis of samples from scenes-of-crime.
To complete the introduction, we looked at the impact of the
internet and World Wide Web, without which much of modern
genetics could not be managed efficiently. In some ways, the growth
and development of internet-based technologies parallels that of DNA
technology itself, and is a good example of how technological and
scientific progress are inextricably linked.

Further Reading

Suggestions for further reading include books, commentary, technical notes,

opinion articles, formal reviews and primary literature, appropriate to each
chapter. Some are classic papers that helped define the subject; others are
general personal reflections, and some have quite a challenging level of detail.
Where possible, articles have been selected as open access, with the URL and
digital object identifier (DOI) listed.

Berg, P. (2001). The Nobel Prize interview. Interview by J. Rose. URL [www
.nobelprize.org/prizes/chemistry/1980/berg/interview/].

ISTUDY
 14 1 INTRODUCTION

Cohen, S. N. (2013). DNA cloning: a personal view after 40 years. Proc. Natl.
Acad. Sci. U. S. A., 110 (39), 15521–9. URL [www.pnas.org/doi/full/10.1073/
pnas.1313397110]. DOI [https://doi.org/10.1073/pnas.1313397110].
Fredrickson, D. S. (1991). Asilomar and recombinant DNA: the end of the
beginning. In: K. E. Hannah, editor. Biomedical Politics. National Academy
Press, Washington, DC; pp. 258–98. URL [www.ncbi.nlm.nih.gov/books/
NBK234217/].

Websearch

People

Four people who played a major role in gene manipulation

becoming a reality – have a look at what they did. They are:
Werner Arber, Daniel Nathans and Hamilton Smith (Nobel Prize 1978),
and Paul Berg (Nobel Prize 1980).

Places

What has Asilomar State Beach in California got to do with genetic

engineering?

Processes

The process of publishing information on the web is something that

affects us all. How do we sift out what is accurate and truthful
from what is not? Pick a topic that you are interested in (not
necessarily biological) and look to see how it is presented by the
types of website listed in Section 1.4. Evaluate critically.

Reflections

What topics in this chapter have you found most challenging?

Look for resources that help to illustrate the key points.

ISTUDY
 Genetic engineering

is an is also known as

requires four
basic steps

enabling generation of gene cloning

technology DNA fragments recombinant DNA technology
with molecular cloning
genetic modification
that involves
joining to a
sub-disciplines
vector or carrier
cutting, modifying has applications in
molecule arose from
such as and joining DNA
molecules basic science
introduction into
genomics using microbial and molecular
a host cell
proteomics biotechnology genetics
bioinformatics used for
DNA profiling various types of
enzymes selection of and was first achieved
forensics medicine
desired
ancient DNA
sequence
such as when recombinant
but raises some complex DNA was generated

type II restriction DNA for use in a

enzymes ligase personal, societal, by
range of
ethical and legal questions
applications
Paul Berg in 1972 at
can be used for Stanford University

Concept Map 1

ISTUDY
 Chapter 2 Summary
Learning Objectives
When you have completed this chapter, you will be able to:
r Outline elements of the philosophy of science
r Describe the scope and ethos of the scientific method
r Identify the infrastructure and support requirements of
research
r Discuss types of data and their evaluation
r Summarise key events, people and places in the history of DNA
research

Key Words
Aristotle, William Whewell, Isaac Newton, Robert Hooke, Louis
Pasteur, Albert Szent-Györgyi, Nobel Prize, Thomas Huxley,
Charles Darwin, Jacob Bronowski, George Wald, iterative,
François Jacob, descriptive, empirical, experimental, hypotheses,
hypothesis-driven, hypothesis-free, genome-wide association
studies (GWAS), classical, reductionist, Occam’s razor, emergent
properties, systems biology, deductive, inductive, consensus
sequence, hypothetico-deductive, Karl Popper, falsifiable,
objective, subjective, data, quantitative, qualitative, valid,
reliable, accuracy, precision, variables, controls, negative
controls, positive controls, independent variable, dependent
variable, confounding variables, biological variation, uncertainty,
error bars, mean, confidence limits, standard error, bias,
unconscious bias, selection bias, confirmation bias, Maurice
Wilkins, primary literature, secondary literature, tertiary
literature, ‘publish or perish’, basic science, pure science,
fundamental science, applied science, technology, paradigm shift,
Thomas Kuhn, geocentric, heliocentric, Newtonian mechanics,
quantum mechanics, analogue, digital, step-changes, paradigm
nudges.

ISTUDY
 Chapter 2

The Story of DNA

In this chapter, we will look in a little more detail at how the science
of DNA has evolved and developed over the past 160 years. Although
this is a long time in terms of human lifespan, it is only a fraction of
the history of science as a recorded discipline. The Ancient Greek
philosopher and polymath Aristotle, who lived in the fourth century
BCE, is often considered the first scientist (although he did not know
the term, as it was first used by William Whewell in 1833). Aristotle
was the first to study biology systematically, so in some ways, we can
trace the history of natural sciences back some 2 400 years. As we saw It took only 50 years to progress
in Chapter 1, DNA technology is a relatively young area of DNA from determining the structure
science, with the 20 years from 1953 to 1973 a time of intense activity of DNA to working out the
sequence of the human genome.
that answered the fundamental questions about DNA structure and
function, and paved the way for the creation of rDNA and the discov-
eries that followed. The next 30 years led to the sequence of the
human genome, and we are now firmly in the post-genomic era. So
how did we get to this point?

2.1 How Science Works

The phrase standing on the shoulders of giants has a long history, but
perhaps its most famous iteration in science is by Isaac Newton (later
Sir Isaac) who, in a 1676 letter to Robert Hooke, wrote ‘If I have seen
farther, it is by standing on the shoulders of giants.’ All progress in
science depends on evaluation and extension of the work of others,
often in small incremental steps, and this is one of the most important
elements of science across the range of disciplines. Louis Pasteur
alluded to this when he stated that ‘chance favours the prepared
mind’, and the biochemist Albert Szent-Györgyi, who was awarded
the Nobel Prize in 1937, said that ‘research is to see what everybody
has seen and think what nobody has thought’. These two statements
provide a good baseline for our look at science; knowing the work of
others is vital if an unsolicited observation is to be recognised as
something potentially significant, and creative thinking is required
to push science forward.

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 18 2 THE STORY OF DNA
Science can be considered as
having a conversation with the
natural world, using the tools of
curiosity and common sense.
Science is not often a simple
linear process, but is a complex
web of ideas, discussions,
experiments and interactions.
ISTUDY
Science as an extension of common sense is also a theme that can
be helpful as a mental backstop when things get convoluted. Thomas
Huxley (a great supporter of his contemporary Charles Darwin) stated
that science is ‘nothing but trained and organised common sense’, and
Jacob Bronowski’s 1951 classic book The Common Sense of Science is still
relevant today. Finally, George Wald, in his 1967 Nobel Prize lecture,
gave what is perhaps the most evocative description of science; it is
worth quoting the opening paragraph here in full:
I have often had cause to feel that my hands are cleverer than my head.
That is a crude way of characterizing the dialectics of experimentation.
When it is going well, it is like a quiet conversation with Nature. One
asks a question and gets an answer, then one asks the next question and
gets the next answer. An experiment is a device to make Nature speak
intelligibly. After that, one only has to listen.
Source: © The Nobel Foundation 1967.
Science is not clear-cut and tidy; as we will see later, it involves a
complex and sometimes messy journey, often using many approaches.
It is certainly not the straightforward process-defined linear sequence
of steps that simple models portray. The ‘conversation with Nature’
phrase reflects this very well – a conversation will ebb and flow, go
down unpredicted avenues, sometimes excite and sometimes not, and
often be frustrating. Science is the same, and for this reason, the back-
and-forth nature of scientific investigation is an example of an
iterative approach. Another helpful concept of how science works
was first proposed by the 1965 Nobel Prize winner François Jacob
who distinguished ‘day science’ from ‘night science’. He thought of
day science as the formal, hypothesis-based approach carried out in
the workplace by scientists who have a goal that can be defined, tested
and evaluated. In contrast, night science is an unconstrained, free-
thinking, creative approach that Jacob called ‘a sort of workshop of
the possible’. As with many aspects of science, these two approaches
are not mutually exclusive, but mutually supportive.
A few more terms are needed if we are to make sense of science as
a process. Many are presented as pairs, with what might be considered
opposite or mutually exclusive meanings: only a brief outline of the
following terms is given here; a useful exercise would be to have a
look at some more extensive definitions of these terms. Be prepared
for some head-scratching and even confusion!
A descriptive approach is characterised by observing, recording
and classifying, whilst an empirical or experimental approach is a
form of investigative science that constructs hypotheses (sing. hypoth-
esis) to try to explain and predict the behaviour of a system. However,
hypothesis testing is not necessarily part of all experimental science,
and the terms hypothesis-driven and hypothesis-free science can be
used. With the advent of genome sequencing, hypothesis-free investi-
gations can be carried out using techniques like genome-wide associ-
ation studies (GWAS) to look for patterns in large data sets. This is
 2.1 HOW SCIENCE WORKS 19

one example of how modern biology has changed significantly over

the past 20 years or so.
In genetics teaching, one perennial question is whether to begin
with a classical or a reductionist approach. The classical route starts
from the Mendelian principles of transmission genetics (what we
might call the ‘behaviour’ of genes), whereas the reductionist
approach would begin with the structure of DNA, genes and
genomes. In practice, both approaches are valid and sensible. The
same applies to classical/reductionist approaches to genetics research
where the topic under investigation will determine which method-
ology is required. DNA sequence data are perhaps the ultimate
reductionist output; studying population genetics needs a different
approach and produces different data sets. Reductionist methodology
is sometimes associated with the principle defined by Occam’s razor, It is usually sensible to consider
in which the simplest explanation for a given circumstance is pre- the simplest explanation for a
ferred. However, there is a danger in taking a reductionist approach scientific question; natural
systems often (but not always)
too far, as biological systems are hierarchical and demonstrate the
avoid generating redundant
concept of emergent properties, where complex systems are built complexity.
from simpler components. The aim of systems biology is to integrate
the elements of a system and try to make sense of how it works as a
whole. The reductionist and systems biology approaches are some-
times seen as antithetical (as are classical, cf. reductionist genetics,
and ‘day’ and ‘night’ science). This tension is not a particularly
helpful view; both approaches are needed and should complement
each other in reaching valid conclusions about a particular topic. An
interesting, yet unsurprising, paradox is that the ultimate reduction-
ist output (a genome sequence) is proving to be the essential baseline
resource that is enabling integrative investigation of how the genome
actually works.
Let’s assume that we have now selected our topic and set the
parameters within which our investigation will be conducted. What
other factors should we be aware of? The form of reasoning that is
used in science is important conceptually but may not always be so in
practice, as the design of an investigative system (if done correctly) Both deductive and inductive
should have considered any potential conflict. Two forms that are reasoning may be needed when
most often cited are deductive and inductive reasoning. Deductive designing a scientific study and
evaluating the outcome.
reasoning takes accepted premises and reaches (deduces) valid conclu-
sions from these premises, whilst inductive reasoning takes a set of
specific observations and reaches (induces) a generalised conclusion.
A more digestible description is that deductive is sometimes called a
top-down approach, moving from the general to the particular, whereas
inductive is a bottom-up approach that moves from the particular to the
general. In practice, it is sometimes hard to determine which parts of
an investigation use which method, but many parts of experimental
science are based on deductive reasoning, as this enables sequential
refinement of an investigation using the question/answer cycle noted
by Wald. However, inductive reasoning also has a role to play – the
identification of a consensus sequence for a particular part of a gene
is one example where looking at a range of specific sequences across a

ISTUDY
 20 2 THE STORY OF DNA
Experimental science relies on
gathering evidence to support or
refute the hypothesis under
investigation, and confirmation
of outcomes by colleagues who
replicate the experiments.
Most experimental science
involves gathering quantitative
data for evaluation.
ISTUDY
number of genes or organisms leads to a generalisation (consensus)
that sequence X usually has base sequence Y, with some variation.
The combination of forming a hypothesis and testing predictions
using a deductive approach is often called the hypothetico–deductive
process, and is considered a central tenet of the scientific method.
A hypothesis should be able to be tested repeatedly by different
scientists; in the words of Karl Popper, it should be falsifiable.
Trying to disprove an interpretation of a particular phenomenon
is just as important in science as marshalling support; gathering
evidence (supportive or otherwise) is the key part of the process.
Related to this, you may come across the terms objective
and subjective, particularly when noting that scientists must be
objective and remove any trace of subjectivity when conducting
research. This is undoubtedly desirable, but we again must remem-
ber that a hypothesis proposed by an individual will inevitably be
set within his or her frame of reference, and so will, to some extent,
be subjective. The ideal of completely objective practice is not
always attainable in the experimental life sciences, and thus aware-
ness of the possibility of subjectivity is important. In our final con-
sideration of the basis of the scientific process, we will look at
some of the important parts of actually doing science – what tools
scientists use, how they behave at a personal level and how they
communicate.
Science is based on evaluation of information, often described as
data. In a technical sense, data are (the term is a plural!) made up of
single pieces of information (a datum) that identifies, describes or
measures a particular part of a system. Scientists also talk about
quantitative and qualitative data sets, where things are measured to
generate quantitative data, or described to generate qualitative data.
Although as ever the demarcation can often be blurry, most experi-
mental science will produce quantitative data for analysis. In
gathering and evaluating quantitative data sets, there are several
factors that need to be considered. Is the measurement valid (does it
measure what it is trying to measure?) and reliable (does the means of
measurement remain consistent in application?). The ‘means of meas-
urement’ might be a simple device like a ruler, or a more complex
machine or a multi-step process. Clearly a ruler has intrinsically a
high level of reliability (although its use may generate unreliable
measurements), whereas a machine may require routine and frequent
servicing and calibration if it is to remain reliable. Related to validity
and reliability, accuracy and precision are important in gathering
data. Accuracy refers to how close a measurement is to the ‘true’
value, whilst precision is a reflection of how close repeated measure-
ments are to each other, or how much variation there is in a set of
measurements. Trying to identify and control validity, reliability,
accuracy and precision is always an interesting exercise in
experimental design.
 2.1 HOW SCIENCE WORKS 21

What do we actually measure? Assuming the issues around valid-

ity, etc. have been addressed, we are often still faced with a complex
experimental system that has many different attributes or variables.
Controlling variables is essential in science, as this provides a way of
‘isolating’ a particular part of the system for investigation. At its
simplest, all variables, apart from the one under investigation, would
be kept constant. Often this is difficult to achieve, and some care is
needed when interpreting results. Using various controls is important
in establishing cause and effect, with both negative controls (absence
of test treatments) and positive controls (treatments known to gener- Identifying and controlling the
ate the test outcome) being used in a single experiment. The variable range of variables that might
that the investigator adjusts or sets as a benchmark is the independ- influence an experimental system
ent variable, and the variable under test is the dependent variable is a key part of the scientific
(i.e. its value depends on the response to the independent variable). method.
Getting in the way are confounding variables, which can affect both
independent and dependent variables and, as the name suggests, can
add noise or confusion to an outcome if not controlled.
Having accounted for all of the elements of experimental design
listed above, we might reasonably expect data sets to be consistent,
and to provide an accurate and valid assessment of the intended
target under investigation. However, no measuring system can be
completely accurate, precise and reliable at all times. Add to that
the inherent biological variation found in living systems and their
component parts, and we will always generate data that have a Quantitative data sets may be
level of uncertainty. This is defined as variation around an already processed using a range of
measured value, and is often quantified by using statistical statistical methods; this approach
can help to make sense of
methods such as error bars (for graphical presentation) and/or
complex data but will not
calculating the mean, confidence limits and standard error (this alleviate any errors in
takes us into the whole area of statistical analysis and will not be experimental design.
described here in detail).
We have so far examined the characteristics of science as a way
of investigating a topic. What of the scientist? The personal attri-
butes of scientists are many and varied. A certain level of intellec-
tual and technical ability is, of course, needed, coupled with a
highly developed sense of curiosity and the desire to understand
how the world around us works. Add to that a dogged determin-
ation, capacity for hard work, an open-minded and honest
approach and care and attention to detail when carrying out
experiments and recording results, and we have some idea of the
basic requirements. Objectivity and freedom from bias (and being
alert to subjective influences) are also expected. Bias comes in a
number of forms, both innate and learnt, and is a complex topic.
Ideological, cultural and political influences can all shape how a Care must be taken to be aware
person views a subject, and unconscious bias can be difficult to of, and avoid, subjectivity and
recognise. Other forms relevant to how scientists work are selec- bias when evaluating
experimental results.
tion bias and confirmation bias, which can affect how experi-
ments are set up and evaluated.

ISTUDY
 22 2 THE STORY OF DNA

The essence of what a scientist does was put very well by Maurice
Wilkins, who shared the 1962 Nobel Prize with Watson and Crick.
When asked ‘What do you actually do?’, his response was: ‘The main
thing in my kind of scientific work is to be able to fiddle with a thing,
go on fiddling with it, and fiddle, fiddle until everyone else has
given up.’
Communication is a major part of how science works, and yet is
sometimes underplayed in simple models of the scientific method.
Whilst it is feasible to design and carry out an experiment, reach a
valid conclusion and then not tell anyone about it, this would be
counterproductive. If unpublicised, the results would not become part
of the body of knowledge about the subject, and thus would not be
available to help the overall progress in the discipline. Scientists are
usually not so reticent, and are almost always keen to discuss, present
and publish their findings as widely as possible. There may be a
constraint on some types of work being published (highly sensitive
government-backed research in some areas is one example), but
unless there is a legal or other compelling reason not to, the aim is
to have results published, critiqued, replicated and ultimately
endorsed by the scientific community. Publication is also vital for
building a personal profile and career, and for attracting finance to
support research.
Communication mechanisms can involve a range of events and
activities:
r Direct communication with peers
r Presenting results at small-scale meetings or seminars
r Presentations at larger-scale national or international conferences
r Submitting results or data to a repository such as a sequence
database
r In-house publications such as institute or company websites
r Publication in the scientific literature (peer-reviewed or
commissioned)
Published work in science usually fits into one of three categories –
primary, secondary or tertiary scientific literature. Results of
experiments and investigations are published in journals that are
part of the primary literature. Conference proceedings that include
novel results, technical reports and research theses also form part
of the primary literature. Secondary literature includes review
articles and books that collate and evaluate primary sources for a
topic. These are broader than original research articles and are very
useful as authoritative accounts of the literature in the subject.
Tertiary literature include textbooks and derivative works such as
dictionaries and encyclopedias (this book is part of the tertiary
literature).
Overall, publication is an accepted marker of scientific success and
standing, although this can bring its own difficulties. There is a
dynamic that is often called ‘publish or perish’, alluding to the

ISTUDY
 2.1 HOW SCIENCE WORKS 23

pressure to publish widely and frequently to ensure that a visible and

respected profile, or perhaps a perceived competitive advantage, is
maintained. This can lead to a tendency to overpublish, from both
scientists and publishers, and many consider this to be a self-
sustaining and problematic development in science publishing.
Although it is not feasible to count the number of scientific journals
accurately, estimates set this at some 30 000 or so, with several mil-
lion papers published annually. This is a good example of how some-
thing that seems simple (count the number of papers published) can
essentially be impossible (think through how validity, reliability,
accuracy and precision might come into play here). Even trying to
keep up to date in a narrow specialism can be problematic, and would
be impossible without IT-based access and search tools.

2.1.1 A Simple Model for the Scientific Method

There is a fairly standard model for the scientific process, variants of
which can be found in innumerable books, science magazines and
websites. Most are based on the hypothetico-deductive process, with
some variation in the number of stages listed. One version is shown in
Fig. 2.1. This shows a process with six defined stages, often carried out
sequentially. The observation can be a simple visual event, or can arise
from a sophisticated technical process involving complex equipment.
Asking a question and constructing a hypothesis usually follow (the

Exte rnal Fig. 2.1 A simple model for the

scientific method. The process
involves a series of steps beginning
n

with an observation and

io

va tion Que progressing through to a

at

er s ti
li c

conclusion, which may be negative

s o
b
Pub

or inconclusive and lead to

n
O

amendment of the observation or

restatement of the question/
Conclusion

Internal hypothesis. Not all stages may

Hypothesis

occur for all investigations, or they

? may be carried out in a different

order. At all stages, internal and
external influences (scientific
literature, discussion, data
processing) can be used to evaluate
and inform. Eventually a consensus
s

xp conclusion forms and leads to

y si
l eri publication of the research.
Ana ment

ISTUDY
 24 2 THE STORY OF DNA
Research requires continual
internal and external questioning
and discussion, covering a range
of topics from day-to-day detail
to high-level strategic planning.
The demarcation of basic
science, applied science and
technology is often imprecise
and may actually be an
unhelpful concept.
Several factors influence how the
scientific process is supported
and enabled.
ISTUDY
‘what if’ question). Gathering data, either by observation or experi-
ment, enables analysis and evaluation of the results, and leads to a
conclusion. The conclusion may be negative or unhelpful, and require
a rethink of the question or the methodology. At all stages, there will
be processing of information in the scientist’s own mind (internal) and
interaction with other scientists and the scientific literature (exter-
nal), much iterative thinking and talking, often a lot of repetition,
problem-solving (why did my restriction enzyme not work?) and mini-
hypothesis testing (if I add Y, instead of Z, will my restriction enzyme
now work?). This ‘day science’ raises more questions, which are some-
times clear and well defined, but which can be rather hazy and
speculative. This is usually where the creative and inspirational parts
of ‘night science’ come to the fore (although this may, of course, occur
in the middle of the afternoon whilst waiting for a reaction to finish!).
And so the ‘conversation with Nature’ continues.
This fairly simple model is not incorrect, and it does show, to some
extent, how the scientific process can be defined. It does not, however,
describe the reality of how science works at the discipline or subject
level. There has been an active debate about the failings of this type of
model for almost as long as DNA has been studied, with many more
sophisticated versions being proposed. We could look at some of
these, but instead I want to broaden the view a little and consider
the framework within which scientists work, with its attendant
opportunities and caveats.
2.1.2 A More Realistic Model for How Science Works
The lines between basic science (often called pure or fundamental
science), applied science and technology are often blurry, and to
maintain this division has become almost redundant. An example is
genome science; this depends on the technological developments that
produced sequencing machines, and there are legitimate questions as
to whether sequencing a gene or genome is pure or applied science.
There are some scientists for whom these distinctions are important,
and who consider that ‘pure’ science should remain untainted by
application or commercialisation. The reality is that in practice, these
definitions don’t matter, and any organisation that does scientific
research may be involved in all three areas concurrently in an inter-
dependent and interdisciplinary setting.
To construct our more realistic model, we will separate the process
of doing the science (as outlined in Fig. 2.1) from the infrastructure
and environment needed to support the activity, and look at the life
science sector primarily. There are several factors that we need to
consider:
r Type of institution (university, research institute, company)
r Funding source(s) and recurrent availability/stability
r Location and links to other facilities and services
 2.2 DNA: A BIOGRAPHICAL TIMELINE 25

Internal External Fig. 2.2 Factors impacting on a

research laboratory. Laboratories
INPUTS Funding can vary in size and may be located
Staff
Permanent scientists Public/government within a larger organisational unit.
Technical support Private sector Core business is where the
Administrative Foundations scientific methodology and process
Research students Charities happen. A range of internal and
Post-doc/fellowships Laboratory external factors provide inputs that
Visiting/short-term Supply chain need to be managed. Outputs may
Core business Lab equipment be publications, data, services
Lab consumables or products.
Specialist chemicals
Infrastructure Logistics
Location Support functions
Building
Links
Basic lab equipment
Universities
Specialist equipment
Research institutions
IT network
Hospitals
Storage/distribution
OUTPUTS Companies

r Staffing – permanent, short-term contract, students, technical,

support
r Supply chain for equipment and consumables
r Distribution network for products (if needed)
r Ideological, cultural, social and political landscape (if relevant)

These factors apply across the sector, albeit at different levels. A small
research group in a university will operate on a completely different
scale than a multinational biotechnology company, but the basic
requirements are broadly similar. Our more realistic model is shown
in Fig. 2.2.

2.2 DNA: A Biographical Timeline

The story of DNA may be unique in terms of the biological systems

studied and the people involved, but it reflects how science works and
has parallels in many other disciplines. Early discoveries helped set
Increasingly sophisticated
the scene for more detailed investigations, often involving experi- experiments and procedures
ments that were relatively simple in a technical sense but were ele- require the development of
gant in their design and enabled Wald’s ‘conversation with Nature’ in new technologies.
a fairly direct way. As more knowledge was gained, the techniques
had to evolve to enable this to continue, and thus molecular biology
has a co-dependence on technology that is clearly illustrated by a
number of the developments since the early-1970s. To tell the story
of DNA, we will look at some of the key discoveries on its biographical
timeline, shown in Fig. 2.3.

ISTUDY
 Mendel investigates transmission of characters in peas 1865

1869 Meischer discovers DNA

1879 Mitosis described by Flemming

Correns, de Vries and von Tschermak

1900 rediscover Mendel’s experiments
1902 Garrod studies the inheritance of alkaptonuria

1909 The word ‘gene’ is coined by Johannsen

Morgan establishes the ‘fly room’ and works out
1911 that genes are carried on chromosomes

Sturtevant constructs the first genetic linkage map 1913

1919 The word ‘biotechnology’ is used by Ereky

Griffith demonstrates bacterial transformation 1928

Beadle and Tatum establish the

1941 ‘one gene one enzyme’ hypothesis

Avery, MacLeod and McCarty show that

DNA is the ‘transforming principle’ 1944

Hershey and Chase confirm that Franklin takes the famous ‘photo 51’ of the X-ray
DNA is the genetic material 1952 diffraction pattern of the B-form of DNA

Watson and Crick determine the

1953 structure of the Double Helix

1955 Tjio confirms human chromosome number 1955 Kornberg isolates DNA polymerase
1956 Ingram establishes the cause of sickle cell disease

Meselson and Stahl show that DNA

replication is semi-conservative 1958
1959 Lejeune shows Down syndrome is caused by trisomy-21

1961 Guthrie develops test for phenylketonuria in newborn babies 1961 Jacob and Monod show how the lac operon is regulated
Brenner, Jacob and Meselson find that mRNA carries the
genetic code from DNA in protein synthesis
Nirenberg, Holley and Khorana decipher the genetic code 1966

1967 Weiss and Richardson purify DNA ligase

1968 Arber, Nathans and Smith discover restriction enzymes

1970 Smith and Nathans isolate type II restriction enzymes

1971 Cetus, the first biotechnology company, established

1972 Berg generates the first rDNA

1973 Asilomar I conference on rDNA risks

Jaenish and Mintz develop transgenic mice 1974

1975 Asilomar II conference and moratorium
1976 The biotechnology company Genentech is formed
Web activity: find
out what the key Roberts and Sharp discover split genes 1977 Rapid DNA sequencing developed by Sanger and Gilbert
DNA-related
discoveries were
in the year that 1982 GenBank database is established Birnster and Palmiter use rat growth
you were born. 1982 Genentech rDNA insulin approved
1982 hormone gene to create ‘supermouse’

Fig. 2.3 DNA timeline. Source: Background image by Robert Brook / Corbis / Getty Images.

ISTUDY
 1983 Huntington disease marker mapped to cs4
1983 Transgenic tobacco plant is generated 1983 Mullis develops the polymerase chain reaction

Jeffreys develops DNA profiling 1985 1985 Discovery of zinc finger nucleases
1986 rDNA vaccine approved for hepatitis B 1986 Positional cloning of chronic granulomatous gene
1989 The cystic fibrosis CFTR gene is identified
Human Genome Project begins 1990 First human gene therapy treatment for SCID

Calgene's Flavr Savr GM tomato goes on sale 1994

1995 H. influenzae genome sequenced

1996 S. cerevisiae genome sequenced 1996 Dolly the sheep cloned

1998 C. elegans genome sequenced
2000 Drosophila and Arabidopsis genomes sequenced
2001 Human genome first draft

Mouse is first mammalian genome to be sequenced 2002 2002 Plasmodium genome sequenced

2003 Glo-Fish GM pet fish on sale 2003 Human Genome Project completed

2004 Rat genome high-quality sequence completed

2005 Chimpanzee genome sequenced 2005 Rice genome sequenced

New DNA sequencing technologies developed

2007

2008 1000 Genomes project launched

First comprehensive analysis of

cancer genomes published
2009

2010 Neanderthal genome sequence published 2010 Venter creates first synthetic life form

2011 Discovery of TALENs

Doudna and Charpentier use CRISPR to edit genomes 2012 100000 Genomes project launched

Esvelt proposes using CRISPR

2014 to spread traits in a gene drive

A major controversy develops as a scientist claims

to have edited the genome of newborn twins 2018

The CRISPR baby scientists are convicted and

jailed for carrying out illegal medical practice
2019 Prime editing makes single-strand-cut editing a possibility

2020 DNA technology plays a major role in managing the

SARS-CoV-2 pandemic by developing clinical
diagnostic tests,whole-genome sequencing for
2021 CRISPR-edited tomato goes on sale 2021 variant analysis and vaccines

A genetically modified pig heart is transplanted

into a patient; he dies after 2 months with pig 2022 T2T completes a gapless reference human genome
cytomegalovirus a possible cause

ISTUDY
Another random document with
no related content on Scribd:
1st Brigade, because it had endured more of the dust, and more of
the delay from casual stoppages and accidents, which always
happens in the rear of a long column. Alten carried the survivors—
the 1st and 3rd battalions Rifle Brigade, 1/43rd, and 1st Caçadores
—as far as Yanzi, and then turned them down the road to the
Bidassoa, which is screened by woods. The French were taken
wholly by surprise: the Rifle battalions carried the knoll above the
bridge, and the bridge itself, without much difficulty. The 43rd and
Caçadores spread themselves out on the slope to their right and
opened fire on the hurrying mass below them.
‘At twilight,’ wrote a captain of the 43rd whose narrative was
quoted by Napier, but is well worth quoting again, ‘we overlooked the
enemy within a stone’s throw, and from the summit of a precipice:
the river separated us: but the French were wedged in a narrow
road, with rocks enclosing them on one side and the river on the
other. Such confusion took place among them as is impossible to
describe. The wounded were thrown down in the rush and trampled
upon: the cavalry drew their swords and endeavoured to charge up
the pass to Echalar[1035], the only opening on their right flank. But the
infantry beat them back, and several of them, horse and man, were
precipitated into the river. Others fired up vertically at us, while the
wounded called out for quarter, and pointed to the numerous soldiers
borne on the shoulders of their comrades on stretchers composed of
the branches of trees, to which were looped great-coats clotted with
gore, or blood-stained sheets taken from houses to carry their
wounded—on whom we did not fire[1036].’
The officer commanding in the French rear finally got out a
battalion behind a stone wall, whose fire somewhat covered the
defiling mass. All the bolder spirits ran the gauntlet through the zone
of fire and escaped up the road to Echalar[1037]. The weak, the
wounded, and the worn-out surrendered to the leading troops of the
4th Division, who now closed in on them. About 1,000 prisoners
were made, largely soldiers of the train and other non-combatants,
but including stragglers from nearly every division in Soult’s army.
There was no pursuit—the Light Division troops could not have
stirred a step: the 4th Division were almost as weary after a long
day’s hunt. The casualties of both had been absurdly small—3
officers and 45 men in Cole’s regiments, 1 officer and 15 men in
Alten’s. The Spanish battalion engaged in the afternoon must, of
course, have lost on a very different scale during its highly creditable
operations; but its casualties have, unluckily, not been recorded. The
French may have had 500 killed and wounded, and 1,000 prisoners
—but this was the least part of their loss—the really important thing
was that thousands of men were scattered in the hills, and did not
rally to their eagles for many days. That night Soult’s army was not
only a demoralized, but a much depleted force.
Wellington was, not unnaturally, dissatisfied with the day’s work.
‘Many events,’ he wrote to Graham, ‘turned out for us unfortunately
on the 1st instant, each of which ought to have been in our favour:
we should have done the enemy a great deal more mischief than we
did during his passage down this valley[1038].’ For one of these things,
Alten’s late arrival, he was himself mainly responsible[1039]: for the
others—Longa’s and Barcena’s strange failure to detach more
troops to help the regiment of Asturias at Yanzi[1040], and Dalhousie’s
late arrival with the 7th Division—which never got into action or lost a
man—he was not.
It must be confessed, however, that Wellington’s intentions on
August 1st are a little difficult to follow. One would have supposed
that he would have devoted his main attention to a direct attempt to
smash up Soult’s main body—but he never allotted more than the
4th, 7th, and Light Divisions to that task—while he had obviously
another idea in his head, dealing with a larger scheme for the
destruction of the enemy. At 9.30, when he had occupied
Santesteban, he sent orders to Byng, then at Elizondo, to bid him to
march at once on the Pass of Maya, and to throw an advanced party
into Urdax, on the French side of the defile. At the same time Hill, at
Irurita, is desired to follow Byng, occupy Elizondo, and—if his troops
can bear the strain—advance even to the Pass of Maya. Should this
prove possible, Byng, when relieved by Hill, should descend into
France as far as Ainhoue on the Nivelle. The 2nd Division, and the
Portuguese division attached to it, would follow him next morning.
Hopes are expressed in the dispatch that Pakenham and the 6th
Division—last heard of at Eugui on the 31st—would be in a position
to combine their operations with those of Hill[1041].
 This descent into the valley of the Nivelle from the Maya pass
must surely have been imagined with the idea of encircling the whole
French army at Echalar, for Ainhoue was well in Soult’s rear, and
troops placed there could cut him off from the direct road to
Bayonne. On the same evening Wellington was dictating a dispatch
to Graham telling him that he hoped to be at Maya next morning, and
beyond the frontier: Graham was therefore to prepare to cross the
Bidassoa and attack Villatte with his and Giron’s full force, including
cavalry and guns, leaving St. Sebastian blockaded by the necessary
minimum detachment[1042]. Meanwhile Alten, Dalhousie, and Cole
were to mass in front of the enemy’s position at Echalar. The only
possible meaning that can be drawn from these orders, when read
together, is that Wellington had now developed the complete
encircling scheme for Soult’s destruction, which he had not thought
out on July 31.
But the scheme was never put into execution. On August 2 Byng
was halted at Maya, Graham received no order to pass the
Bidassoa, and all that was done was to execute an attack on Echalar
—attended with complete success, it is true, but only resulting in
pushing Soult back towards the Nivelle, where there was no
intercepting force waiting to waylay him. Somewhere between 8 p.m.
on the 1st and dawn on the 2nd Wellington, for reasons which he did
not avow to his staff or his most trusted lieutenants, gave up the
greater game, which had in it immense possibilities: for Soult had no
longer an army that could fight—as events at Echalar were to prove
a few hours later. Minor causes for this great piece of self-restraint
can be cited. One was that Hill and Pakenham turned out not to be
within easy supporting distance of Byng, so that the dash into Soult’s
rear could not be executed with a sufficient force. If this operation
were not carried out, Graham’s became useless. Certainly a
reflection on the extremely depleted condition of the 2nd Division,
which had suffered such heavy losses at Maya, Beunza, and the
Venta de Urroz, helped to deter Wellington from using this exhausted
force for his main stroke. Writing to Graham two days later, he
mentioned its condition as a cause of delay, along with a general
dearth of musket ammunition and shoes. But the main reason, as we
shall see later, lay rather in the higher spheres of European politics,
and the explanation was reserved for the Minister of War in London
alone. Next morning no general attack was delivered: Wellington did
not go to Maya, but merely joined the 4th and Light Divisions on the
Bidassoa. The great scheme was stillborn, and never took shape.
Soult had gathered the wrecks of his army on the range of
mountains behind Echalar, where the Spanish-French boundary line
runs. The cavalry had been at once packed off to the rear. The
infantry took up a position. Clausel’s three divisions were in the
centre—Vandermaesen holding the village with a company, and with
his main body—certainly not 2,000 men that day—across the road
on the slope above. Conroux’s division was on Vandermaesen’s
right: Taupin’s, in reserve to both the others, on the crest where the
frontier runs. Reille’s corps—still more depleted than Clausel’s,
continued the line westward—Lamartinière’s division next to
Conroux’s, as far as the Peak of Ivantelly: Maucune’s—the most
dilapidated unit in a dilapidated army—holding the ground beyond
the Ivantelly, with a flank-guard out on its right watching the road
from Vera to Sarre. D’Erlon’s divisions—still by far the most intact
units of the whole command, were on Clausel’s left, prolonging the
line eastward and holding a commanding position on the mountain
side as far as the Peaks of Salaberry and Atchuria. It is doubtful
whether the Marshal had 25,000 men in line that day in his eight
divisions—not because the remainder were all casualties or
prisoners, but because the long retreat, with its incessant scrambling
over mountain sides, had led to the defection of many voluntary and
still more involuntary stragglers. The former were scattered over the
country for miles on every side, seeking for food and for ‘a day off’—
the latter had dropped behind from sheer exhaustion: there had been
no regular distribution of rations since July 29th, and the first convoy
of relief had been captured by Byng at Elizondo on the 31st.
Wellington had only 12,000 men available in front of a very
formidable position—hills 1,500 or 1,800 feet high, with the peaks
which formed the flank protection rising to 2,100 or 2,300. Under
ordinary circumstances an attack would have been insane.
Moreover, his troops were almost as way-worn as those of Soult: the
Light and 4th Divisions had received no rations since the 30th, and
the latter had lost a good third of its strength in the very heavy
fighting in which it had taken the chief part between the 25th and the
30th. But their spirits were high, they had the strongest confidence in
their power to win, and they were convinced that the enemy was ‘on
the run’—in which idea they were perfectly right.
The plan of attack was that the 4th and 7th Divisions should
assail the enemy’s centre, on each side of the village of Echalar,
while the Light Division turned his western flank. This involved long
preliminary marching for Alten’s men, despite of their awful fatigues
of the preceding day. They had to trudge from the bridge of Yanzi
and Aranaz to Vera, where they turned uphill on to the heights of
Santa Barbara, a series of successive slopes by which they
ascended towards the Peak of Ivantelly and Reille’s flank. Just as
they began to deploy they got the first regular meal that they had
seen for two days: ‘The soldiers were so weak that they could hardly
stand; however, our excellent commissary had managed to overtake
us, and hastily served out half a pound of biscuit to each individual,
which the men devoured in the act of priming and loading just as
they moved off to the attack[1043].’ The morning was dull and misty—a
great contrast to the blazing sunshine of the preceding day, and it
was hard to get any complete view of the position—clouds were
drifting along the hills and obscuring parts of the landscape for many
minutes at a time. This chance put Wellington himself in serious
danger for a moment—pushing forward farther than he knew, with a
half-company of the 43rd to cover him, he got among the French
outposts, and was only saved by the vigilance of his escort from
being cut off—he galloped back under a shower of balls—any one of
which might have caused a serious complication in the British
command—it is impossible to guess what Beresford would have
made of the end of the campaign of 1813.
While the Light Division was developing the flank attack, the front
attack was already being delivered—somewhat sooner than
Wellington expected or intended. The plan had been for the 4th
Division to operate against the French right—the 7th against their
centre and left centre. Cole, however, was delayed in getting forward
by the immense block of French débris along the narrow defile from
Sumbilla to the bridge of Yanzi. ‘For two miles there were scattered
along the road papers, old rugs, blankets, pack-saddles, old bridles
and girths, private letters, hundreds of empty and broken boxes,
quantities of entrenching tools, French clothes, dead mules, dead
soldiers, dead peasants, farriers’ tools, boots and linen, the boxes of
M. le Général Baron de St. Pol[1044] and other officers, the field
hospital of the 2nd Division (Darmagnac’s), and all sorts of things
worth picking up—which caused stoppage and confusion[1045].’
Now the 7th Division did not follow the spoil-strewn river road, but
cut across from Sumbilla towards Echalar, over the same hill-tracks
which Clausel’s divisions had taken on the preceding afternoon,
when they escaped from the pursuit of Cole’s skirmishers. Hence it
chanced that they arrived in front of Echalar on a route where the
enemy was keeping no good watch, long before the 4th Division
came up from the bridge of Yanzi and the Vera cross-roads. The
mists on the hills had kept them screened—as Clausel complains in
his report. Lord Dalhousie now carried out a most dangerous
manœuvre—a frontal attack on an enemy in position by a series of
brigades arriving at long intervals—without any co-operation having
been sought or obtained from the troops known to be on his left.
‘Bravery and success,’ as a Light-Division neighbour observed,
‘certainly far exceeded judgement or utility[1046].’ What led the
commander of the 7th Division to this astounding escapade was the
obvious unpreparedness of the enemy. ‘We caught them,’ he wrote,
‘cooking above, and plundering below in the village. I thought it best
to be at them instantly, and I really believe Barnes’s brigade was
among them before their packs were well on[1047].’
The leading troops, and the only ones which really got into action,
were the three battalions (1/6th, 3rd Provisional[1048], and Brunswick-
Oels) of Barnes’s brigade—led by that same fighting general who
had stopped the rout at Maya with two of these same battalions.
Barnes got his line formed, and attacked uphill against the front of
Conroux’s division, long before Inglis’s brigade was ready to follow,
or Lecor’s Portuguese had even got down from the hill path into the
narrow valley of the Sari stream. With such speed and vigour was
the assault delivered, under a frontal fire from Conroux’s men, and a
flank fire from Vandermaesen’s on the right, that Inglis’s brigade,
which was aiming at the village of Echalar, never had the chance of
getting near its enemy. The advancing line suffered severely as it
climbed—nearly 300 casualties—but when it came against the front
of Conroux, and delivered its first volley, the enemy simply melted
away[1049]. As Clausel writes in apology, ‘the resistance ought to have
been greater, and in the ordinary state of the army, that is to say
when a better spirit prevailed, it would never have been possible for
the enemy to establish himself in this fashion on a section of the
main chain of the Pyrenees. This day the morale of the troops was
bad[1050].’ It must be remembered that Conroux’s was the division
which had suffered so heavily in the village of Sorauren both on the
28th and the 30th of July. Several of its battalions were skeletons—
all much thinned. Still there must have been 3,000 men yet present
out of the original 7,000—and they turned and fled before the uphill
attack of 1,800 or less. Nor was this the end of the disaster. Clausel
tried to hurry Vandermaesen’s division to the succour of Conroux’s.
But the manœuvre failed: the French General says that Conroux’s
flying troops ran in upon Vandermaesen’s, that confusion followed,
and that he was obliged to let the whole mass roll back to seek
shelter with Taupin’s division in the reserve line[1050].
At this moment the leading brigade of the 4th Division, that of
Ross, at last appeared on Dalhousie’s left, and began to skirmish
with Lamartinière’s line[1051]: demonstrations began—probably by
Lecor’s Portuguese—against the front of Maransin[1052], D’Erlon’s
right-hand division. But these were of no importance in comparison
with the effect of the turning of Reille’s end of the line by the Light
Division. This was almost as startling in effect as Barnes’s dealings
with the French centre. Having reached the front on the heights of
Santa Barbara, below the Peak of Ivantelly, Alten brushed aside
Maucune’s feeble skirmishing line, and let loose five companies of
the Rifle Brigade supported by four of the 43rd against the
dominating peak of the Ivantelly, the most prominent feature of the
French position. Clouds swept down along the hills at this moment,
and the supporting companies lost sight of the front line. ‘An invisible
firing commenced, and it was impossible to ascertain which party
was getting the better of the fight: the combatants were literally
contending in the clouds[1053].’ But when the 43rd came up, they
found that the rifle companies had dislodged the 2nd Léger,
Lamartinière’s flank regiment, from the precipitous crest, and were in
full possession of it. As they lost only 1 officer and 26 men in taking a
most formidable peak, it is clear that the enemy’s resistance must
have been very ineffective[1054].
 All was now confusion on the French right wing—Reille speaks of
himself as wandering about in a fog with three battalions of
Lamartinière’s, and meeting no one save the brigadier Montfort, who
was bringing up a mere 200 men to try to reinforce the troops on the
Ivantelly. The soldiers were profiting by the mist to go off to the rear,
and could not be kept together. ‘On se tirailla faiblement, et nos
troupes se retirèrent sur la route de Sarre.’ With his centre smashed
in, and his right dispersed, Soult could do nothing but retire. The
remains of Clausel’s and Reille’s divisions fell back on the road to
Sarre. D’Erlon, who had never been attacked, could not use this
route, but made his way along the crest of the mountains to
Zagaramurdi, Urdax, and Ainhoue. What would have been his fate if
he had found there not the picquet which (by Wellington’s order)
Byng had thrown forward to Urdax, but the whole of Byng’s and Hill’s
troops blocking his retreat—as would have been the case if the great
scheme drawn up on the night of August 1 had been carried out? But
the Pass of Maya had not been utilized that day, and D’Erlon had
only to drive away 50 men.
There was no pursuit: if there had been it would seem that the
whole French army would have broken up, for it had shown itself this
day no longer able to fight. ‘The spirit not only of the men but of the
officers,’ wrote Reille next morning, ‘has been very bad during these
last days. The absolute want of food must be the excuse for this
state of things.’ The condition of the army was deplorable—
Maucune’s division showed less than 1,000 men holding together.
The 1st Line of Vandermaesen’s had precisely 27 men with the
eagle—yet had lost only 4 officers and 193 men in action—where
were the remaining 400? Other units could show a few hundred men
but absolutely or practically no officers—the 55th Line of Conroux
had lost every one of 13 officers, the 51st Line of Darmagnac 12 out
of 17, the 34th Léger of Maucune 30 out of 35; these were
exceptionally hard cases, but in all the divisions save those of Foy
and Abbé (the least engaged during the short campaign), the
proportion was appalling. For the infantry of the whole army it was
420 casualties among 1,318 officers present. Soult wrote to Clarke
on the last day of the campaign: ‘I deceived myself in the strangest
way when I told your Excellency that the troops had their morale
intact, and would do their duty. I mistook the sentiment of shame for
their recent disaster (Vittoria) for that of steadfastness. When tested,
they started with one furious rush, but showed no power of
resistance.... Since I first entered the service I have seen nothing like
this. It reminded me of the behaviour of the levy en masse of 1792.
The spirit of these troops must be terribly broken to have permitted
them to behave in this fashion. One general told me that he had
overheard men remarking, when we were near Pampeluna, that they
had better not fight too hard, because it would be preferable to get
back to the frontier rather than to be led off into the middle of
Spain[1055].’ These were cruel and spiteful words—the army had
fought with excellent courage, till after July 30th it had convinced
itself that the Marshal had got wrong with all his calculations, that the
game was up—that they were being taken on a wild goose chase
without rations in the most desolate and rocky region of Europe. But
by August 2 Soult was not far out in his statement—on that day the
greater part of his army was a spent force. If Wellington had resolved
to pursue with vigour, he could have pushed it as far as he pleased.
Perhaps the great encircling scheme with which his mind dallied for
a few hours on the night of August 1 might have resulted in its
surrender or complete dispersion.
This was not to be. On August 3 Wellington halted, and
commenced to rearrange his troops on much the same principles,
and in much the same positions, that he had selected before. He
wrote to Graham next day that he was perfectly well aware of the
objection to taking up a defensive position in the Pyrenees, but that
an advance was too risky[1056]. It was not so much the prospect of the
wastage of his troops, even in successful operation, that deterred
him, though this was the point on which he insisted in his letter to
Graham, but the general political situation of Europe. The eternal
Armistice of Plässwitz was still holding up operations in Germany: it
was still possible that the Allied Sovereigns might make a selfish
peace with Napoleon, and permit the Emperor to expand Soult’s
army ad infinitum with sudden reinforcements. What would then
become of the Anglo-Portuguese host, even if it had won its way not
only to Bayonne but to Bordeaux? All this he had considered, and
wrote to the Secretary for War in Whitehall that ‘as for the immediate
invasion of France, from what I have seen of the state of
negotiations in the North of Europe, I have determined to consider it
only in reference to the convenience of my own operations.... If
peace should be made by the Powers of the North, I must
necessarily withdraw into Spain.’ No advance, however tempting the
opportunity, should be made until he was certain that war had
recommenced in Saxony[1057]. Meanwhile, ‘Soult will not feel any
inclination to renew his expeditions—on this side at least.’
So the army settled down to hold once more the line of the
Pyrenees, and to resume the siege of St. Sebastian. On August 3
the Head-quarters were once more at Lesaca, as they had been on
July 25—how many things had happened in the interval! Hill and the
2nd Division, now rejoined by the long-missing brigade of Byng, as
also Silveira’s Portuguese, were in the Maya passes once more. The
3rd Division was holding the Roncesvalles defiles, the 4th and 7th
were at Echalar, the 6th in the Alduides (replacing Campbell’s
Portuguese in that remote valley); the Light Division lay on the
heights opposite Vera. Morillo’s Division was in the Bastan behind
Hill, the part of the Army of Reserve of Andalusia which O’Donnell
had carried to the front, when he left the rest before Pampeluna, was
at the moment near the bridge of Yanzi. The remainder of that army
and Carlos de España were continuing the blockade of Pampeluna.
The total losses of the Allies during the nine days’ ‘Campaign of
the Pyrenees’ had amounted to slightly over 6,400 officers and men
for the English and Portuguese[1058]. The Spanish loss could not add
over 600 more—at Sorauren it was 192; Morillo’s casualties at
Altobiscar, and those of the regiment of Asturias at Yanzi bridge are
the only unknown quantities, and can hardly have reached 400. The
distribution among divisions and corps was odd—the 3rd and Light
Division had practically negligible casualties: the former under 120,
the latter under 50. The main stress fell on the 2nd and 4th Divisions,
the former with 2,000 British and 350 Portuguese casualties, the
latter with 1,400 and 300 respectively. But it must be remembered
that the 2nd Division had (including Byng) four brigades, the 4th
Division only three. Every unit in both was severely tried; the most
terrible return was that of the 1/92nd with its 26 officers and 445 men
killed, wounded, and missing. But this was a strong battalion of 750
bayonets, and I am not sure that the 284 casualties of the 20th and
the 296 casualties of the 3/27th, both in the 4th Division, do not
represent almost as great a proportional loss, as these were both
much smaller corps.
The 6th Division was engaged on two days only, at the two
battles of Sorauren, and had the appreciable number of 450 British
and 370 Portuguese casualties. The 7th Division fought on three
days—at second Sorauren, at the Combat of the Venta de Urroz,
and at Echalar, and three of its battalions also saved the day at
Maya—with a loss in all of 750 British but only 60 Portuguese. Lastly,
we must name the two Portuguese brigades of Silveira’s division, of
which Campbell’s fought at Sorauren, Da Costa’s at Beunza, with a
loss to the former of 350 and to the latter of 280 men. The general
fact emerges that the 2nd and 4th Divisions lost between them 4,350
men out of the total 7,000—no other division had so many as 1,000
casualties.
The official list of French losses is not quite complete, as it
includes only the infantry and cavalry units—but such casualties as
may have been suffered by the general staff, the artillery (very little
engaged) sappers, and train can have added comparatively little to
the total, though a good many men of the train were taken at Yanzi
on August 1. The figures given work out to 12,563—1,308 killed,
8,545 wounded, and 2,710 prisoners. The last-named total should
probably be brought up to 3,000 in order to include individuals of the
non-combatant corps captured in the retreat. The divisions suffered
in very unequal measure: Foy and Abbé got off very lightly, because
the one was practically not engaged at first Sorauren, nor the latter
at Maya—they lost respectively only 550 and 750 men.
Vandermaesen’s and Maucune’s Divisions—each a small unit of only
4,000 men, had the crushing losses of 1,480 and 1,850 respectively,
and were dissolved when the campaign was over, and re-formed to a
large extent with battalions drawn from the Bayonne reserve.
Darmagnac and Conroux each lost well over 2,000 men out of 7,000
—their divisions having been terribly cut up, the one at Maya the
other at Sorauren. Maransin and Taupin each return over 1,000 men
lost out of 6,000; Lamartinière just under 1,000 out of 7,000. The
cavalry, barely engaged and quite useless, had only 67 casualties to
report.
But to get at Soult’s fighting strength on the last day of the
retreat, it is not sufficient merely to deduct the 12,563 casualties from
the 59,000 which took the field in Navarre. The eight divisions
collected on the 2nd August were short not only of their own
casualties, but of Foy’s division, which had gone off on an eccentric
line of retreat, as had several smaller detachments[1059]. But the great
deficit was that of at least 8,000 or 10,000 stragglers, who rejoined at
their leisure during the next ten days. The cavalry and artillery had all
gone to the rear, and it is doubtful if the last stand at Echalar was
made with so many as 25,000 weary infantry. If the Marshal had
been faced with an opponent who had no political arrière pensée to
hold him back, he would have been doomed to absolute destruction.
Only a wreck of his army would have escaped, and Wellington might
have driven the remnants as far as he pleased—even to the gates of
Bordeaux.
But the British General had made his ‘great refusal’ on the night
of August 1st-2nd, and had resolved that the tempting scheme for
invading France, which had flitted before his eyes for a few evening
hours, must be postponed. He would risk nothing till he should get
certain information that the war had begun again in Germany. And as
the news of the rupture of the Armistice did not reach him till
September 3rd, he was condemned to another month of waiting. He
turned back to his old policy of June—St. Sebastian and Pampeluna
must be reduced. When they should have fallen, it would be time
enough to see whether the general European situation had made the
invasion of France a feasible enterprise.
 APPENDICES

I
BRITISH LOSSES AT THE SIEGE OF BURGOS
SEPTEMBER 20-OCTOBER 21, 1812

Sept. 20th, storm of the Hornwork of St. Michael. Total casualties 421
(of whom the 42nd lost 2 officers and 33 men killed, 5 officers and
164 men wounded = 204; the 79th lost 5 men killed, 2 officers
and 32 men wounded = 39; the Portuguese lost 3 officers and 17
men killed, 5 officers and 88 men wounded = 113; the other 67
casualties were divided among the remaining battalions of the 1st
Division).
Sept. 21st, casualties 42; Sept. 22nd, 39; Sept. 23, 158 (of which 76
in the Guards Brigade and 44 in the K.G.L. brigade of the 1st
Division, and 29 among the Portuguese).
Sept. 24th, casualties 39; Sept. 25th, 38; Sept. 26th, 32; Sept. 27th,
53; Sept. 28th, 26; Sept. 29th, 9; Sept. 30th, 29; Oct. 1st, 11; Oct.
2nd, 29; Oct. 3rd, 9; Oct. 4th, 16; Oct. 5th, 224 (of these 224,
which really belong to the storm of the outer enceinte on the
evening of Oct. 4th, the 2/24th had 68 casualties, no other
battalion more than 15).
Oct. 6th, casualties (French sortie of night of 5th) 142; Oct. 7th, 33;
Oct. 8th (2nd French sortie in which 133 in the K.G.L. Brigade
were killed or wounded), 184; Oct. 9th, 18; Oct. 10th, 61; Oct.
11th, 24; Oct. 12th, 17; Oct. 13th, 9; Oct. 14th, 3; Oct. 15th, 18;
Oct. 16th, 12; Oct. 17th, 18; Oct. 18th, 48; Oct. 19th (the last
 assault), 170 (of whom 85 in the Guards Brigade and 84 in the
K.G.L. Brigade of the 1st Division).
Oct. 20th, 47; Oct. 21st, 9.

General Total: 24 officers and 485 men killed

68 ” ” 1,445 ” wounded
42 ” missing = 2,064
 II
THE FRENCH ARMIES IN SPAIN: OCTOBER 15,
1812
[From the return in the Archives Nationales, Paris.]

I. ARMY OF THE SOUTH. MARSHAL SOULT

Officers. Men.
1st Division, Conroux: 9th Léger, 24th, 96th Line (each 3
batts.): 3 battalions Marine Troops 203 5,615
2nd Division, Barrois[1060]: 8th Line, 16th Léger, 51st, 54th
Line (each 3 batts.) 201 4,801
3rd Division, Villatte: 27th Léger, 63rd, 94th, 95th Line (each
3 batts.) 209 5,888
4th Division, Leval: 32nd, 43rd Line (4 batts. each), 55th,
58th Line (3 batts. each) 259 7,794
5th Division, D’Erlon[1061]: 12th Léger (4 batts.), 45th Line (3
batts.), 28th Léger, 88th Line (2 batts. each) 202 5,016
6th Division, Daricau: 21st Léger, 100th Line (3 batts. each),
64th, 103rd Line (2 batts. each) 187 4,308
Cavalry Division, Perreymond: 2nd Hussars, 5th, 10th, 21st,
27th Chasseurs, 7th Lancers (in all, 20 squad.) 144 2,349
Cavalry Division, Digeon: 2nd, 4th, 14th, 17th, 26th, 27th
Dragoons (in all, 20 squadrons) 148 2,956
Cavalry Division, Pierre Soult: 5th, 12th, 16th, 21st
Dragoons (14 squadrons) 121 1,712
Artillery, and Artillery Train and Park 122 3,511
Engineers and Sappers 17 851
Gendarmerie and Équipages Militaires 9 592
État-Major 313 —
Total present 1,816 45,393
Gross total of Army including 64 officers and 1,904 men 1,940 53,590
 detached, and 60 officers and 6,293 men in hospital:
II. ARMY OF THE CENTRE
Officers. Men.
The King’s French Guards: no figures given, but about 2,500
Darmagnac’s Division: 28th and 75th Line (3 batts. each),
2nd Nassau and Baden (2 batts. each), Frankfort (1 batt.) 199 5,039
Palombini’s Division: 4th and 6th Italian Line (2 batts. each),
2nd Italian Léger (3 batts.), Dragoons of Napoleon and
two batteries 142 3,050
Casapalacios’ Spanish Division (3 batts., 3 squadrons) 167 1,263
Treillard’s Cavalry Division: 13th, 18th, 19th, 22nd
Dragoons, Westphalian chevaux légers, Nassau
chasseurs 114 1,679
Artillery, Engineers, and Train 12 596
Various detachments 12 403
Total present 646 14,530
Gross total of Army, including État-Major 55 officers, 32
officers and 655 men detached, and 14 officers and 1,900
men sick, is 747 17,075
III. ARMY OF PORTUGAL. GENERAL SOUHAM
Officers. Men.
1st Division, Foy: 6th Léger (1 batt.), 39th, 69th, 76th Line
(2 batts. each) 151 3,492
2nd Division, Clausel: 25th Léger, 27th, 50th, 59th Line (2
batts. each) 130 4,506
3rd Division, Taupin [late Ferey], 31st Léger, 26th, 70th Line
(2 batts. each), 47th Line (3 batts.) 184 6,064
4th Division, Sarrut: 2nd and 4th Léger, 36th Line (2 batts.
each) 130 4,000
5th Division, Maucune: 15th, 66th, 82nd, 86th Line (2 batts.
each) 165 5,097
6th Division, Pinoteau [late Brenier]: 17th Léger, 65th Line
(2 batts. each), 22nd Line (1 batt.) 87 2,730
7th Division, Bonté [late Thomières]: 1st and 62nd Line (2
batts. each), 101st Line (1 batt.) 109 2,425
8th Division, Chauvel [late Bonnet]: 118th, 119th, 120th Line
(2 batts. each), 122nd Line (3 batts.) 180 4,437
 (Each infantry division includes its artillery and train.)
Cavalry Division, Curto: 3rd Hussars, 13th, 14th, 22nd,
26th, 28th Chasseurs 115 2,048
Cavalry Division, Boyer: 6th, 11th, 15th, 25th Dragoons 70 1,303
Horse Artillery and train attached to cavalry 5 292
Artillery Reserve and Park 37 1,822
Engineers 7 274
Gendarmerie and Équipages Militaires 33 1,102
Attached to the Army of Portugal—
Cavalry Brigade, Merlin, of the Army of the North, 1st
Hussars, 31st Chasseurs 53 693
Infantry Brigade, Aussenac, detached from the Bayonne
Reserve: 3rd and 105th Line (2 batts. each), 64th,
100th, 103rd (1 batt. each) 110 3,308
Total present 1,566 43,860
Gross total of Army with 150 officers and 4,574 men
detached, and 53 officers and 11,113 men in hospital: 1,769 59,547
61,316
IV. ARMY OF ARAGON AND VALENCIA. MARSHAL SUCHET
Officers. Men.
1st Division, Musnier: 1st Léger (3 batts.), 114th Line (2
batts.), 121st Line (3 batts.), Neapolitans (2 batts.) 180 5,403
2nd Division, Harispe: 7th, 44th, 116th Line (2 batts. each) 104 4,011
3rd Division, Habert: 14th, 16th Line (2 batts. each), 117th
Line (3 batts.) 158 4,817
Cavalry Division, Boussard: 14th Cuirassiers, 24th
Dragoons, 4th Hussars, 1st Neapolitan Chasseurs 91 1,831
Artillery and train 21 858
Engineers 11 411
Gendarmerie and Équipages Militaires 6 529

Troops attached to the Army of Aragon

Reille’s Division: 10th Line (4 batts.), 81st (3 batts.), 9th bis
of Hussars, artillery 147 4,393
Severoli’s Division: 1st Italian Line and 1st Italian Léger (2
batts. each), 5th Léger (French) (1 batt.), and Italian
artillery 151 3,758
Brigade from Catalonia: 3rd Léger, 11th, 20th Line (2 batts. 130 3,719
 each), 1st Italian Chasseurs
Total present 1,038 30,980
Gross total of Army, including 131 officers and 3,884 men
detached, and 24 officers and 4,287 men in hospital: 1,193 39,151
V. ARMY OF THE NORTH. GENERAL CAFFARELLI
Officers. Men.
Division Abbé: 5th Léger (3 batts.), 10th Léger, 3rd, 52nd,
105th Line (2 batts. each), 20th Dragoons, and artillery 219 6,378
Division Vandermaesen: 34th Léger (1 batt.), 34th, 40th
Line (3 batts. each), 113th, 130th Line (2 batts. each), 4th
Suisse (1 batt.), 6 bataillons de marche = 18 battalions in
all 388 12,197
Brigade Dumoustier: 4 batts. Young Guard, and 2 of
National Guards 100 3,976
Cavalry Brigade Laferrière: Lancers of Berg (2 squadrons),
15th Chasseurs (3 squadrons), Gendarmerie (6
squadrons) 37 1,625
Government of Navarre (garrison of Pampeluna)
detachments 45 1,644
Government of Biscay, all régiments de marche and
detachments 264 9,431
Government of Castile (garrison of Santoña) detachments 43 1,342
Total present 1,151 36,465
Gross total of Army, including 41 officers and 5,176 men in
hospital, is 1,192 41,641
VI. ARMY OF CATALONIA. GENERAL DECAEN
Officers. Men.
Division Quesnel (at Puycerda), 102nd Line (2 batts.), 116th
Line (1 batt.), and detachments 123 3,502
Division Lamarque: 60th and 79th Line (3 batts. each),
Regiment of Wurzburg, and detachments 137 3,491
Brigade Petit: 67th Line (3 batts.), 32nd Léger (1 batt.) 66 1,553
Division Maurice Mathieu (Barcelona): 115th Line (4 batts.),
18th Léger (2 batts.), 1st of Nassau (2 batts.), and
detachments 185 6,180
Garrisons of Figueras, &c.: 60th, 86th Line, and 32nd Léger
(1 batt. each), and detachments 160 3,646
Garrison of Tarragona (Bertoletti): 20th Line (1 batt.), 7th 63 1,451
Italian Line (1 batt.)
Garrison of Lerida (Henriod): 42nd Line (3 batts.), and
detachments 70 1,639
Brigade Espert (flying column): 5th Line (3 batts.), 23rd
Léger (2 batts.) 89 2,988
Artillery, Sappers, Gendarmes, Équipages Militaires, &c. 55 4,194
Total present, under arms 948 28,514
Gross total of Army, including 107 État-Major, 26 officers
and 369 men detached, and 44 officers and 6,045 men in
hospital, is 1,125 34,928
VII. BAYONNE RESERVE
Officers. Men.
All consisting of detachments or régiments de marche 172 7,072

Adding 134 detached and 600 in hospital, gross total is: 7,978
Total Army of Spain, therefore, on October 15 amounts to:
7,516 officers, 206,814 men present with the colours
326 ” 11,520 ” detached
236 ” 35,414 ” in hospital.
General Gross Total = 8,185 officers and 253,748 men.