Note: Supplemental materials are not guaranteed with Rental or Used book purchases.
Purchase Benefits
What is included with this book?
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 |
Table of Contents provided by Publisher. All Rights Reserved. |