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From Genes to Genomes : Concepts and Applications of DNA Technology,9780470683859
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From Genes to Genomes : Concepts and Applications of DNA Technology



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The latest edition of this highly successful textbook introduces the key techniques and concepts involved in cloning genes and in studying their expression and variation. The new edition features: Increased coverage of whole-genome sequencing technologies and enhanced treatment of bioinformatics. Clear, two-colour diagrams throughout. A dedicated website including all figures. Noted for its outstanding balance between clarity of coverage and level of detail, this book provides an excellent introduction to the fast moving world of molecular genetics.

Table of Contents

Prefacep. xiii
From Genes to Genomesp. 1
Introductionp. 1
Basic molecular biologyp. 4
The DNA backbonep. 4
The base pairsp. 6
RNA structurep. 10
Nucleic acid synthesisp. 11
Coiling and supercoilinp. 11
What is a gene?p. 13
Information flow: gene expressionp. 15
Transcriptionp. 16
Translationp. 19
Gene structure and organisationp. 20
Operonsp. 20
Exons and intronsp. 21
Refinements of the modelp. 22
How to Clone a Genep. 25
What is cloning?p. 25
Overview of the proceduresp. 26
Extraction and purification of nucleic acidsp. 29
Breaking up cells and tissuesp. 29
Alkaline denaturationp. 31
Column purificationp. 31
Detection and quantitation of nucleic acidsp. 32
Gel electrophoresisp. 33
Analytical gel electrophoresisp. 33
Preparative gel electrophoresisp. 36
Restriction endonucleasesp. 36
Specificityp. 37
Sticky and blunt endsp. 40
Ligationp. 42
Optimising ligation conditionsp. 44
Preventing unwanted ligation: alkaline phosphatase and double digestsp. 46
Other ways of joining DNA fragmentsp. 48
Modification of restriction fragment endsp. 49
Linkers and adaptorsp. 50
Homopolymer tailingp. 52
Plasmid vectorsp. 53
Plasmid replicationp. 54
Cloning sitesp. 55
Selectable markersp. 57
Insertional inactivationp. 58
Transformationp. 59
Vectors based on the lambda bacteriophagep. 61
Lambda biologyp. 61
In vitro packagingp. 65
Insertion vectorsp. 66
Replacement vectorsp. 68
Cosmidsp. 71
Supervectors: YACs and BACsp. 72
Summaryp. 73
Genomic and cDNA Librariesp. 75
Genomic librariesp. 77
Partial digestsp. 77
Choice of vectorsp. 80
Construction and evaluation of a genomic libraryp. 83
Growing and storing librariesp. 86
cDNA librariesp. 87
Isolation of mRNAp. 88
cDNA synthesisp. 89
Bacterial cDNAp. 93
Screening libraries with gene probesp. 94
Hybridizationp. 94
Labelling probesp. 98
Steps in a hybridization experimentp. 99
Screening procedurep. 100
Probe selection and generationp. 101
Screening expression libraries with antibodiesp. 103
Characterization of plasmid clonesp. 106
Southern blotsp. 107
PCR and sequence analysisp. 108
Polymerase Chain Reaction (PCR)p. 109
The PCR reactionp. 110
PCR in practicep. 114
Optimisation of the PCR reactionp. 114
Primer designp. 115
Analysis of PCR productsp. 117
Contaminationp. 118
Cloning PCR productsp. 119
Long-range PCRp. 121
Reverse-transcription PCRp. 123
Quantitative and real-time PCRp. 123
SYBR Greenp. 123
TaqManp. 125
Molecular beaconsp. 125
Applications of PCRp. 127
Probes and other modified productsp. 127
PCR cloning strategiesp. 128
Analysis of recombinant clones and rare eventsp. 129
Diagnostic applicationsp. 130
Sequencing a Cloned Genep. 131
DNA sequencingp. 131
Principles of DNA sequencingp. 131
Automated sequencingp. 136
Extending the sequencep. 137
Shotgun sequencing; contig assemblyp. 138
Databank entries and annotationp. 140
Sequence analysisp. 146
Identification of coding regionp. 146
Expression signalsp. 147
Sequence comparisonsp. 148
DNA sequencesp. 148
Protein sequence comparisonsp. 151
Sequence alignments: Clustalp. 157
Protein structurep. 160
Structure predictionsp. 160
Protein motifs and domainsp. 162
Confirming gene functionp. 165
Allelic replacement and gene knockoutsp. 166
Complementationp. 168
Analysis of Gene Expressionp. 169
Analysing transcriptionp. 169
Northern blotsp. 170
Reverse transcription-PCRp. 171
In situ hybridizationp. 174
Methods for studying the promoterp. 174
Locating the promoterp. 175
Reporter genesp. 177
Regulatory elements and DNA-binding proteinsp. 179
Yeast one-hybrid assaysp. 179
DNase I footprintingp. 181
Gel retardation assaysp. 181
Chromatin immunoprecipitation (ChIP)p. 183
Translational analysisp. 185
Western blotsp. 185
Immunocytochemistry and immunohistochemistryp. 187
Products from Native and Manipulated Cloned Genesp. 189
Factors affecting expression of cloned genesp. 190
Transcriptionp. 190
Translation initiationp. 192
Codon usagep. 193
Nature of the protein productp. 194
Expression of cloned genes in bacteriap. 195
Transcriptional fusionsp. 195
Stability: conditional expressionp. 198
Expression of lethal genesp. 201
Translational fusionsp. 201
Yeast systemsp. 204
Cloning vectors for yeastsp. 204
Yeast expression systemsp. 206
Expression in insect cells: baculovirus systemsp. 208
Mammalian cellsp. 209
Cloning vectors for mammalian cellsp. 210
Expression in mammalian cellsp. 213
Adding tags and signalsp. 215
Tagged proteinsp. 215
Secretion signalsp. 217
In vitro mutagenesisp. 218
Site-directed mutagenesisp. 218
Synthetic genesp. 223
Assembly PCRp. 223
Synthetic genomesp. 224
Protein engineeringp. 224
Vaccinesp. 225
Subunit vaccinesp. 225
DNA vaccinesp. 226
Genomic Analysisp. 229
Overview of genome sequencingp. 229
Strategiesp. 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 technologiesp. 239
De novo sequence assemblyp. 239
Repetitive elements and gapsp. 240
Analysis and annotationp. 242
Identification of ORFsp. 243
Identification of the function of genes and their productsp. 250
Other features of nucleic acid sequencesp. 251
Comparing genomesp. 256
BLASTp. 256
Syntenyp. 257
Genome browsersp. 258
Relating genes and functions: genetic and physical mapsp. 260
Linkage analysisp. 261
Ordered libraries and chromosome walkingp. 262
Transposon mutagenesis and other screening techniquesp. 263
Transposition in bacteriap. 263
Transposition in Drosophilap. 266
Transposition in other organismsp. 268
Signature-tagged mutagenesisp. 269
Gene knockouts, gene knockdowns and gene silencingp. 271
Metagenomicsp. 273
Conclusionp. 274
Analysis of Genetic Variationp. 275
Single nucleotide polymorphismsp. 276
Direct sequencingp. 278
SNP arraysp. 279
Larger scale variationsp. 280
Microarrays and indelsp. 281
Other methods for studying variationp. 282
Genomic Southern blot analysis: restriction fragment length polymorphisms (RFLPs)p. 282
VNTR and microsatellitesp. 285
Pulsed-field gel electrophoresisp. 287
Human genetic variation: relating phenotype to genotypep. 289
Linkage analysisp. 289
Genome-wide association studies (GWAS)p. 292
Database resourcesp. 294
Genetic diagnosisp. 294
Molecular phylogenyp. 295
Methods for constructing treesp. 298
Post-Genomic Analysisp. 305
Analysing transcription: transcriptomesp. 305
Differential screeningp. 306
Other methods: transposons and reportersp. 308
Array-based methodsp. 308
Expressed sequence tag (EST) arraysp. 309
PCR product arraysp. 310
Synthetic oligonucleotide arraysp. 312
Important factors in array hybridizationp. 313
Transcriptome sequencingp. 315
Translational analysis: proteomicsp. 316
Two-dimensional electrophoresisp. 317
Mass spectrometryp. 318
Post-translational analysis: protein interactionsp. 320
Two-hybrid screeningp. 320
Phage display librariesp. 321
Epigeneticsp. 323
Integrative studies: systems biologyp. 324
Metabolomic analysisp. 324
Pathway analysis and systems biologyp. 325
Modifying Organisms: Transgenicsp. 327
Transgenesis and cloningp. 327
Common species used for transgenesisp. 328
Control of transgene expressionp. 330
Animal transgenesisp. 333
Basic methodsp. 333
Direct injectionp. 333
Retroviral vectorsp. 335
Embryonic stem cell technologyp. 336
Gene knockoutsp. 339
Gene knock-down technology: RNA interferencep. 340
Gene knock-in technologyp. 341
Applications of transgenic animalsp. 342
Disease prevention and treatmentp. 343
Live vaccine production: modification of bacteria and virusesp. 343
Gene therapyp. 346
Viral vectors for gene therapyp. 347
Transgenic plants and their applicationsp. 349
Introducing foreign genesp. 349
Gene subtractionp. 351
Applicationsp. 352
Transgenics: a coda 353 Glossaryp. 355
Bibliographyp. 375
Indexp. 379
Table of Contents provided by Publisher. All Rights Reserved.

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