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. 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. Preface xiii1 From Genes to Genomes 11.1 Introduction 11.2 Basic molecular biology 41.2.1 The DNA backbone 41.2.2 The base pairs 61.2.3 RNA structure 101.2.4 Nucleic acid synthesis 111.2.5 Coiling and supercoilin 111.3 What is a gene? 131.4 Information flow: gene expression 151.4.1 Transcription 161.4.2 Translation 191.5 Gene structure and organisation 201.5.1 Operons 201.5.2 Exons and introns 211.6 Refinements of the model 222 How to Clone a Gene 252.1 What is cloning? 252.2 Overview of the procedures 262.3 Extraction and purification of nucleic acids 292.3.1 Breaking up cells and tissues 292.3.2 Alkaline denaturation 312.3.3 Column purification 312.4 Detection and quantitation of nucleic acids 322.5 Gel electrophoresis 332.5.1 Analytical gel electrophoresis 332.5.2 Preparative gel electrophoresis 362.6 Restriction endonucleases 362.6.1 Specificity 372.6.2 Sticky and blunt ends 402.7 Ligation 422.7.1 Optimising ligation conditions 442.7.2 Preventing unwanted ligation: alkaline phosphatase and double digests 462.7.3 Other ways of joining DNA fragments 482.8 Modification of restriction fragment ends 492.8.1 Linkers and adaptors 502.8.2 Homopolymer tailing 522.9 Plasmid vectors 532.9.1 Plasmid replication 542.9.2 Cloning sites 552.9.3 Selectable markers 572.9.4 Insertional inactivation 582.9.5 Transformation 592.10 Vectors based on the lambda bacteriophage 612.10.1 Lambda biology 612.10.2 In vitro packaging 652.10.3 Insertion vectors 662.10.4 Replacement vectors 682.11 Cosmids 712.12 Supervectors: YACs and BACs 722.13 Summary 733 Genomic and cDNA Libraries 753.1 Genomic libraries 773.1.1 Partial digests 773.1.2 Choice of vectors 803.1.3 Construction and evaluation of a genomic library 833.2 Growing and storing libraries 863.3 cDNA libraries 873.3.1 Isolation of mRNA 883.3.2 cDNA synthesis 893.3.3 Bacterial cDNA 933.4 Screening libraries with gene probes 943.4.1 Hybridization 943.4.2 Labelling probes 983.4.3 Steps in a hybridization experiment 993.4.4 Screening procedure 1003.4.5 Probe selection and generation 1013.5 Screening expression libraries with antibodies 1033.6 Characterization of plasmid clones 1063.6.1 Southern blots 1073.6.2 PCR and sequence analysis 1084 Polymerase Chain Reaction (PCR) 1094.1 The PCR reaction 1104.2 PCR in practice 1144.2.1 Optimisation of the PCR reaction 1144.2.2 Primer design 1154.2.3 Analysis of PCR products 1174.2.4 Contamination 1184.3 Cloning PCR products 1194.4 Long-range PCR 1214.5 Reverse-transcription PCR 1234.6 Quantitative and real-time PCR 1234.6.1 SYBR Green 1234.6.2 TaqMan 1254.6.3 Molecular beacons 1254.7 Applications of PCR 1274.7.1 Probes and other modified products 1274.7.2 PCR cloning strategies 1284.7.3 Analysis of recombinant clones and rare events 1294.7.4 Diagnostic applications 1305 Sequencing a Cloned Gene 1315.1 DNA sequencing 1315.1.1 Principles of DNA sequencing 1315.1.2 Automated sequencing 1365.1.3 Extending the sequence 1375.1.4 Shotgun sequencing; contig assembly 1385.2 Databank entries and annotation 1405.3 Sequence analysis 1465.3.1 Identification of coding region 1465.3.2 Expression signals 1475.4 Sequence comparisons 1485.4.1 DNA sequences 1485.4.2 Protein sequence comparisons 1515.4.3 Sequence alignments: Clustal 1575.5 Protein structure 1605.5.1 Structure predictions 1605.5.2 Protein motifs and domains 1625.6 Confirming gene function 1655.6.1 Allelic replacement and gene knockouts 1665.6.2 Complementation 1686 Analysis of Gene Expression 1696.1 Analysing transcription 1696.1.1 Northern blots 1706.1.2 Reverse transcription-PCR 1716.1.3 In situ hybridization 1746.2 Methods for studying the promoter 1746.2.1 Locating the promoter 1756.2.2 Reporter genes 1776.3 Regulatory elements and DNA-binding proteins 1796.3.1 Yeast one-hybrid assays 1796.3.2 DNase I footprinting 1816.3.3 Gel retardation assays 1816.3.4 Chromatin immunoprecipitation (ChIP) 1836.4 Translational analysis 1856.4.1 Western blots 1856.4.2 Immunocytochemistry and immunohistochemistry 1877 Products from Native and Manipulated Cloned Genes 1897.1 Factors affecting expression of cloned genes 1907.1.1 Transcription 1907.1.2 Translation initiation 1927.1.3 Codon usage 1937.1.4 Nature of the protein product 1947.2 Expression of cloned genes in bacteria 1957.2.1 Transcriptional fusions 1957.2.2 Stability: conditional expression 1987.2.3 Expression of lethal genes 2017.2.4 Translational fusions 2017.3 Yeast systems 2047.3.1 Cloning vectors for yeasts 2047.3.2 Yeast expression systems 2067.4 Expression in insect cells: baculovirus systems 2087.5 Mammalian cells 2097.5.1 Cloning vectors for mammalian cells 2107.5.2 Expression in mammalian cells 2137.6 Adding tags and signals 2157.6.1 Tagged proteins 2157.6.2 Secretion signals 2177.7 In vitro mutagenesis 2187.7.1 Site-directed mutagenesis 2187.7.2 Synthetic genes 2237.7.3 Assembly PCR 2237.7.4 Synthetic genomes 2247.7.5 Protein engineering 2247.8 Vaccines 2257.8.1 Subunit vaccines 2257.8.2 DNA vaccines 2268 Genomic Analysis 2298.1 Overview of genome sequencing 2298.1.1 Strategies 2308.2 Next generation sequencing (NGS) 2318.2.1 Pyrosequencing (454) 2328.2.2 SOLiD sequencing (Applied Biosystems) 2358.2.3 Bridge amplification sequencing (Solexa/Ilumina) 2378.2.4 Other technologies 2398.3 De novo sequence assembly 2398.3.1 Repetitive elements and gaps 2408.4 Analysis and annotation 2428.4.1 Identification of ORFs 2438.4.2 Identification of the function of genes and their products 2508.4.3 Other features of nucleic acid sequences 2518.5 Comparing genomes 2568.5.1 BLAST 2568.5.2 Synteny 2578.6 Genome browsers 2588.7 Relating genes and functions: genetic and physical maps 2608.7.1 Linkage analysis 2618.7.2 Ordered libraries and chromosome walking 2628.8 Transposon mutagenesis and other screening techniques 2638.8.1 Transposition in bacteria 2638.8.2 Transposition in Drosophila 2668.8.3 Transposition in other organisms 2688.8.4 Signature-tagged mutagenesis 2698.9 Gene knockouts, gene knockdowns and gene silencing 2718.10 Metagenomics 2738.11 Conclusion 2749 Analysis of Genetic Variation 2759.1 Single nucleotide polymorphisms 2769.1.1 Direct sequencing 2789.1.2 SNP arrays 2799.2 Larger scale variations 2809.2.1 Microarrays and indels 2819.3 Other methods for studying variation 2829.3.1 Genomic Southern blot analysis: restriction fragment length polymorphisms (RFLPs) 2829.3.2 VNTR and microsatellites 2859.3.3 Pulsed-field gel electrophoresis 2879.4 Human genetic variation: relating phenotype to genotype 2899.4.1 Linkage analysis 2899.4.2 Genome-wide association studies (GWAS) 2929.4.3 Database resources 2949.4.4 Genetic diagnosis 2949.5 Molecular phylogeny 2959.5.1 Methods for constructing trees 29810 Post-Genomic Analysis 30510.1 Analysing transcription: transcriptomes 30510.1.1 Differential screening 30610.1.2 Other methods: transposons and reporters 30810.2 Array-based methods 30810.2.1 Expressed sequence tag (EST) arrays 30910.2.2 PCR product arrays 31010.2.3 Synthetic oligonucleotide arrays 31210.2.4 Important factors in array hybridization 31310.3 Transcriptome sequencing 31510.4 Translational analysis: proteomics 31610.4.1 Two-dimensional electrophoresis 31710.4.2 Mass spectrometry 31810.5 Post-translational analysis: protein interactions 32010.5.1 Two-hybrid screening 32010.5.2 Phage display libraries 32110.6 Epigenetics 32310.7 Integrative studies: systems biology 32410.7.1 Metabolomic analysis 32410.7.2 Pathway analysis and systems biology 32511 Modifying Organisms: Transgenics 32711.1 Transgenesis and cloning 32711.1.1 Common species used for transgenesis 32811.1.2 Control of transgene expression 33011.2 Animal transgenesis 33311.2.1 Basic methods 33311.2.2 Direct injection 33311.2.3 Retroviral vectors 33511.2.4 Embryonic stem cell technology 33611.2.5 Gene knockouts 33911.2.6 Gene knock-down technology: RNA interference 34011.2.7 Gene knock-in technology 34111.3 Applications of transgenic animals 34211.4 Disease prevention and treatment 34311.4.1 Live vaccine production: modification of bacteria and viruses 34311.4.2 Gene therapy 34611.4.3 Viral vectors for gene therapy 34711.5 Transgenic plants and their applications 34911.5.1 Introducing foreign genes 34911.5.2 Gene subtraction 35111.5.3 Applications 35211.6 Transgenics: a coda 353Glossary 355Bibliography 375Index 379