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Preface	p. xiii
From Genes to Genomes	p. 1
Introduction	p. 1
Basic molecular biology	p. 4
The DNA backbone	p. 4
The base pairs	p. 6
RNA structure	p. 10
Nucleic acid synthesis	p. 11
Coiling and supercoilin	p. 11
What is a gene?	p. 13
Information flow: gene expression	p. 15
Transcription	p. 16
Translation	p. 19
Gene structure and organisation	p. 20
Operons	p. 20
Exons and introns	p. 21
Refinements of the model	p. 22
How to Clone a Gene	p. 25
What is cloning?	p. 25
Overview of the procedures	p. 26
Extraction and purification of nucleic acids	p. 29
Breaking up cells and tissues	p. 29
Alkaline denaturation	p. 31
Column purification	p. 31
Detection and quantitation of nucleic acids	p. 32
Gel electrophoresis	p. 33
Analytical gel electrophoresis	p. 33
Preparative gel electrophoresis	p. 36
Restriction endonucleases	p. 36
Specificity	p. 37
Sticky and blunt ends	p. 40
Ligation	p. 42
Optimising ligation conditions	p. 44
Preventing unwanted ligation: alkaline phosphatase and double digests	p. 46
Other ways of joining DNA fragments	p. 48
Modification of restriction fragment ends	p. 49
Linkers and adaptors	p. 50
Homopolymer tailing	p. 52
Plasmid vectors	p. 53
Plasmid replication	p. 54
Cloning sites	p. 55
Selectable markers	p. 57
Insertional inactivation	p. 58
Transformation	p. 59
Vectors based on the lambda bacteriophage	p. 61
Lambda biology	p. 61
In vitro packaging	p. 65
Insertion vectors	p. 66
Replacement vectors	p. 68
Cosmids	p. 71
Supervectors: YACs and BACs	p. 72
Summary	p. 73
Genomic and cDNA Libraries	p. 75
Genomic libraries	p. 77
Partial digests	p. 77
Choice of vectors	p. 80
Construction and evaluation of a genomic library	p. 83
Growing and storing libraries	p. 86
cDNA libraries	p. 87
Isolation of mRNA	p. 88
cDNA synthesis	p. 89
Bacterial cDNA	p. 93
Screening libraries with gene probes	p. 94
Hybridization	p. 94
Labelling probes	p. 98
Steps in a hybridization experiment	p. 99
Screening procedure	p. 100
Probe selection and generation	p. 101
Screening expression libraries with antibodies	p. 103
Characterization of plasmid clones	p. 106
Southern blots	p. 107
PCR and sequence analysis	p. 108
Polymerase Chain Reaction (PCR)	p. 109
The PCR reaction	p. 110
PCR in practice	p. 114
Optimisation of the PCR reaction	p. 114
Primer design	p. 115
Analysis of PCR products	p. 117
Contamination	p. 118
Cloning PCR products	p. 119
Long-range PCR	p. 121
Reverse-transcription PCR	p. 123
Quantitative and real-time PCR	p. 123
SYBR Green	p. 123
TaqMan	p. 125
Molecular beacons	p. 125
Applications of PCR	p. 127
Probes and other modified products	p. 127
PCR cloning strategies	p. 128
Analysis of recombinant clones and rare events	p. 129
Diagnostic applications	p. 130
Sequencing a Cloned Gene	p. 131
DNA sequencing	p. 131
Principles of DNA sequencing	p. 131
Automated sequencing	p. 136
Extending the sequence	p. 137
Shotgun sequencing; contig assembly	p. 138
Databank entries and annotation	p. 140
Sequence analysis	p. 146
Identification of coding region	p. 146
Expression signals	p. 147
Sequence comparisons	p. 148
DNA sequences	p. 148
Protein sequence comparisons	p. 151
Sequence alignments: Clustal	p. 157
Protein structure	p. 160
Structure predictions	p. 160
Protein motifs and domains	p. 162
Confirming gene function	p. 165
Allelic replacement and gene knockouts	p. 166
Complementation	p. 168
Analysis of Gene Expression	p. 169
Analysing transcription	p. 169
Northern blots	p. 170
Reverse transcription-PCR	p. 171
In situ hybridization	p. 174
Methods for studying the promoter	p. 174
Locating the promoter	p. 175
Reporter genes	p. 177
Regulatory elements and DNA-binding proteins	p. 179
Yeast one-hybrid assays	p. 179
DNase I footprinting	p. 181
Gel retardation assays	p. 181
Chromatin immunoprecipitation (ChIP)	p. 183
Translational analysis	p. 185
Western blots	p. 185
Immunocytochemistry and immunohistochemistry	p. 187
Products from Native and Manipulated Cloned Genes	p. 189
Factors affecting expression of cloned genes	p. 190
Transcription	p. 190
Translation initiation	p. 192
Codon usage	p. 193
Nature of the protein product	p. 194
Expression of cloned genes in bacteria	p. 195
Transcriptional fusions	p. 195
Stability: conditional expression	p. 198
Expression of lethal genes	p. 201
Translational fusions	p. 201
Yeast systems	p. 204
Cloning vectors for yeasts	p. 204
Yeast expression systems	p. 206
Expression in insect cells: baculovirus systems	p. 208
Mammalian cells	p. 209
Cloning vectors for mammalian cells	p. 210
Expression in mammalian cells	p. 213
Adding tags and signals	p. 215
Tagged proteins	p. 215
Secretion signals	p. 217
In vitro mutagenesis	p. 218
Site-directed mutagenesis	p. 218
Synthetic genes	p. 223
Assembly PCR	p. 223
Synthetic genomes	p. 224
Protein engineering	p. 224
Vaccines	p. 225
Subunit vaccines	p. 225
DNA vaccines	p. 226
Genomic Analysis	p. 229
Overview of genome sequencing	p. 229
Strategies	p. 230
Next generation sequencing (NGS)	p. 231
Pyrosequencing (454)	p. 232
SOLiD sequencing (Applied Biosystems)	p. 235
Bridge amplification sequencing (Solexa/Ilumina)	p. 237
Other technologies	p. 239
De novo sequence assembly	p. 239
Repetitive elements and gaps	p. 240
Analysis and annotation	p. 242
Identification of ORFs	p. 243
Identification of the function of genes and their products	p. 250
Other features of nucleic acid sequences	p. 251
Comparing genomes	p. 256
BLAST	p. 256
Synteny	p. 257
Genome browsers	p. 258
Relating genes and functions: genetic and physical maps	p. 260
Linkage analysis	p. 261
Ordered libraries and chromosome walking	p. 262
Transposon mutagenesis and other screening techniques	p. 263
Transposition in bacteria	p. 263
Transposition in Drosophila	p. 266
Transposition in other organisms	p. 268
Signature-tagged mutagenesis	p. 269
Gene knockouts, gene knockdowns and gene silencing	p. 271
Metagenomics	p. 273
Conclusion	p. 274
Analysis of Genetic Variation	p. 275
Single nucleotide polymorphisms	p. 276
Direct sequencing	p. 278
SNP arrays	p. 279
Larger scale variations	p. 280
Microarrays and indels	p. 281
Other methods for studying variation	p. 282
Genomic Southern blot analysis: restriction fragment length polymorphisms (RFLPs)	p. 282
VNTR and microsatellites	p. 285
Pulsed-field gel electrophoresis	p. 287
Human genetic variation: relating phenotype to genotype	p. 289
Linkage analysis	p. 289
Genome-wide association studies (GWAS)	p. 292
Database resources	p. 294
Genetic diagnosis	p. 294
Molecular phylogeny	p. 295
Methods for constructing trees	p. 298
Post-Genomic Analysis	p. 305
Analysing transcription: transcriptomes	p. 305
Differential screening	p. 306
Other methods: transposons and reporters	p. 308
Array-based methods	p. 308
Expressed sequence tag (EST) arrays	p. 309
PCR product arrays	p. 310
Synthetic oligonucleotide arrays	p. 312
Important factors in array hybridization	p. 313
Transcriptome sequencing	p. 315
Translational analysis: proteomics	p. 316
Two-dimensional electrophoresis	p. 317
Mass spectrometry	p. 318
Post-translational analysis: protein interactions	p. 320
Two-hybrid screening	p. 320
Phage display libraries	p. 321
Epigenetics	p. 323
Integrative studies: systems biology	p. 324
Metabolomic analysis	p. 324
Pathway analysis and systems biology	p. 325
Modifying Organisms: Transgenics	p. 327
Transgenesis and cloning	p. 327
Common species used for transgenesis	p. 328
Control of transgene expression	p. 330
Animal transgenesis	p. 333
Basic methods	p. 333
Direct injection	p. 333
Retroviral vectors	p. 335
Embryonic stem cell technology	p. 336
Gene knockouts	p. 339
Gene knock-down technology: RNA interference	p. 340
Gene knock-in technology	p. 341
Applications of transgenic animals	p. 342
Disease prevention and treatment	p. 343
Live vaccine production: modification of bacteria and viruses	p. 343
Gene therapy	p. 346
Viral vectors for gene therapy	p. 347
Transgenic plants and their applications	p. 349
Introducing foreign genes	p. 349
Gene subtraction	p. 351
Applications	p. 352
Transgenics: a coda 353 Glossary	p. 355
Bibliography	p. 375
Index	p. 379
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From Genes to Genomes : Concepts and Applications of DNA Technology

0470683856

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