The ABCs of Gene Cloning

227 Pages·2007·11.87 MB·English

S E C O N D E D I T I O N
The
ABCs
of
Gene
Cloning
Dominic W.S. Wong
The ABCs of Gene Cloning
Dominic W.S. Wong
The sCBA of Gene Cloning
Second Edition
Springer
Library of Congress Control Number: 2005931435
ISBN-10:0-387-28663-2 e-ISBN: 0-387-28679-9
ISBN- :31 978-0387-28663- I
Printed on acid-free paper.
© 2006 Springer Science+Business Media, Inc.
First edition © 1997 by Chapman & Hall
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CONTENTS
Preface
Part One. Fundamentals of Genetic Processes
1. Introductory Concepts 3
1.1 What is DNA and What is a Gene? 3
1.2 What is Gene Cloning? 4
1.3 Cell Organization 5
1.4 Heredity Factors and Traits 6
1.5 Mitosis and Meiosis 9
1.6 Relating Genes to Inherited Trait 10
1.7 Why Gene Cloning? 11
Review 13
2. Structures of Nucleic Acids 15
2.1 3'-0H and 5'-P Ends 15
2.2 Purine and Pyrimidine Bases 16
2.3 Complementary Base Pairing 17
2.4 Writing a DNA Molecule 18
2.5 Describing DNA sizes 19
2.6 Denaturation and Renaturation 19
2.7 Ribonucleic Acid 20
Review 21
Structures of Proteins 23
3.1 Amino Acids 23
3.2 The Peptide Bond 25
3.3 Structural Organization 26
3.4 Postranslational Modification 27
3.5 Enzymes 28
Review 30
The Genetic Process 31
4.1 From Genes to Proteins 31
4.2 Transcription 31
4.3 Translation 32
4.4 The Genetic Code 33
4.5 Why Present a Sequence Using the Coding Strand? 34
4.6. The Reading Frame 35
4.7 DNA Replication 37
4.8 The Replicon and Replication Origin 38
4.9 Relating Replication to Gene Cloning 39
Review 39
Organization of Genes 41
5.1 The Lactose Operon 41
5.2 Control of Transcription 42
5.2.1 The Transcription Start Site and Termination Site 42
5.2.2 When Does Transcription Start or Stop? 44
5.3. Control of Translation 46
5.3.1 Ribosome Binding Site and Start Codon 46
5.3.2 Translation Termination Site 47
5.4 The Tryptophan Operson 47
5.4.1 Co-repressor 48
5.4.2 Attenuation 48
5.4.3 Hybrid Promoters 49
5.5 The Control System in Eukaryotic Cells 49
5.5.1 Transcriptional Control 49
5.5.2 Introns and Exons 51
5.5.3 Capping and Tailing 52
5.5.4 Ribosome Binding Sequence 52
5.5.5 Monocistronic and Polycistronic 52
Review 60
6. Reading the Nucleotide Sequence of a Gene 55
6.1 The E. coli dut gene 55
6.2 The human bgn gene 58
6.2.1 Reading the genomic sequence 58
6.2.2 Reading the cDNA sequence 62
Part Two. Techniques and Strategies of Gene Cloning
7. Enzymes Used in Cloning 69
7.1 Restriction Enzymes 69
7.2 Ligase 70
7.3 DNA Polymerases 71
7.3.1 E. coli DNA Polymerase 1 71
7.3.2 Bacteriophage T4 and T7 polymerase 73
7.3.3 Reverse Transcriptase 74
7.3.4 RNA Polymerase 74
7.4 Phosphatase and kinase 74
Review 75
8. Techniques Used in Cloning 77
8.1 DNA Isolation 77
8.2 Gel Electrophoresis 77
8.2.1 Agarose Gel Electrophoresis 78
8.2.2 Polyacrylamide Gel Electrophoresis 79
8.3 Wester Blot 80
8.4 Southern Transfer 80
8.5 Colony Blot 81
8.6 Hybridization 82
8.7 Immunological Techniques 83
8.8 DNA Sequencing 85
8.9 Polymerase Chain Reaction 87
8.10 Site-Directed Mutagenesis 89
8.11 Non-radioactive Detection Methods 90
Review 91
Cloning Vectors for Introducing Genes into Host Cells 93
9.1 Vectors for Bacterial Cells 93
9.1.1 Plasmid Vecors 93
9.1.2 Bacteriophage Vectors 99
9.1.3 Cosmids 104
9.1.4 Phagemids 105
9.2 Yeast Cloning Vectors 106
9.2.1 The 2|i Circle 106
9.3 Vectors for Plant Cells 108
9.3.1 Binary Vector System 109
9.3.2 Cointegrative Vector System 110
9.3.3 Genetic Markers 111
9.3.4 Plant Specific Promoters 113
9.4 Vectors for Mammahan Cells 114
9.4.1 SV40 Viral Vectors 114
9.4.2 Direct DNA Transfer 116
9.4.3 Insect Baculovirus 117
9.4.4 Retrovirus 121
Review 123
10. Transformation 125
10.1 Calcium Salt Treatment 125
10.2 Electroporation 126
10.3 Agrobacterium Infection 126
10.4 The Biolistic Process 126
10.5 Viral Transfection 127
10.6 Microinjection 127
10.7 Nuclear Transfer 128
Review 129
11. Isolating Genes for Cloning 131
11.1 The Genome Library 131
11.2 The cDNA Library 132
11.3 Choosing The Right Cell Types for mRNA Isolation 134
Review 134
Part Three. Impact of Gene Cloning - Applications in Agriculture
12 Improving Tomato Quality by Antisense RNA 137
12.1 Antisense RNA 137
12.2 A Strategy for Engineering Tomatoes with 139
Review 141
13. Transgenic Crops Engineered with Insecticidal Activity
13.1 Bacillus thuringiensis Toxins 143
13.2 Cloning of the cry gene into cotton plants 143
13.2.1 Modifying the cry gene 144
13.2.2 The Intermediate Vector 144
13.2.3 Transformation hy Agrobacterium 144
Review 145
14. Transgenic Crops Conferred with Herbicide Resistance 147
14.1 Glyphosate 148
14.2 Cloning of the aroA gene 148
Review 149
15. Growth Enhancement in Transgenic Fish 153
15.1 Gene Transfer in Fish 153
15.2 Cloning Salmons with a Chimeric Growth
Hormone Gene 154
Review 155
Part Four. Impact of Gene Cloning - Applications in Medicine
16. Microbial Production of Recombinant Human Insulin 159
16.1 Structure and Action of Insulin 159
16.2 Cloning Human Insulin Gene 160
Review 161
17. Finding Disease-Causing Genes 163
17.1 Genetic Linkage 163
17.1.1 Frequency of Recombination 164
17.1.2 Genetic Markers 165
17.2 Positional Cloning 165
17.2.1 Chromosome Walking 166
17.2.2 Chromosome Jumping 167
17.2.3 Yeast Artificial Chromosome 167
17.3 Exon Amplification 168
17.4 Isolafion of the Mouse Ofte^e Gene 169
Review 170
18. Human Gene Therapy 171
18.1 Physical Chemical Methods 172
18.2 Biological Methods 173
18.2.1 Life Cycle of Retroviruses 173
18.2.2 Construction of a Safe Retrovirus Vector 173
18.3 Gene Treatment of Severe Combined
Immune Deficiency 174
18.4 Therapeutic Vaccines 175
18.4.1 Construction of DNA Vaccines 176
18.4.2 Methods of Delivery 176
Review 176
19. Gene Targeting 179
19.1 Recombination 179
19.2 Replacement Targeting Vectors 180
19.3 Gene Targeting Without Selectable Markers 182
19.3.1 The PCR Method 182
19.3.2 The Double-Hit Method 183
19.3.3 The Cre/7oxP Recombination 183
19.4 Gene Targeting for Xenotransplants 184
Review 186
20. DNA Typing 187
20.1 Variable Number Tandem Repeats 187
20.2 Polymorphism Analysis Using VNTR Markers 188
20.3 Single-locus and Multi-locus Probes 189
20.4 Paternity Case Analysis 190
20.5 Short Tandem Repeat Markers 191
20.5.1 The Combined DNA Index System 192
20.6 Mitochondrial DNA Sequence Analysis 193
Review 196
21. Transpharmers - Bioreactors for Pharmaceutical
Products 197