Table of ContentsPreface xiii 1 From Genes to Genomes 1 1.1 Introduction 1 1.2 Basic molecular biology 4 1.2.1 The DNA backbone 4 1.2.2 The base pairs 6 1.2.3 RNA structure 10 1.2.4 Nucleic acid synthesis 11 1.2.5 Coiling and supercoilin 11 1.3 What is a gene? 13 1.4 Information flow: gene expression 15 1.4.1 Transcription 16 1.4.2 Translation 19 1.5 Gene structure and organisation 20 1.5.1 Operons 20 1.5.2 Exons and introns 21 1.6 Refinements of the model 22 2 How to Clone a Gene 25 2.1 What is cloning? 25 2.2 Overview of the procedures 26 2.3 Extraction and purification of nucleic acids 29 2.3.1 Breaking up cells and tissues 29 2.3.2 Alkaline denaturation 31 2.3.3 Column purification 31 2.4 Detection and quantitation of nucleic acids 32 2.5 Gel electrophoresis 33 2.5.1 Analytical gel electrophoresis 33 2.5.2 Preparative gel electrophoresis 36 2.6 Restriction endonucleases 36 2.6.1 Specificity 37 2.6.2 Sticky and blunt ends 40 2.7 Ligation 42 2.7.1 Optimising ligation conditions 44 2.7.2 Preventing unwanted ligation: alkaline phosphatase and double digests 46 2.7.3 Other ways of joining DNA fragments 48 2.8 Modification of restriction fragment ends 49 2.8.1 Linkers and adaptors 50 2.8.2 Homopolymer tailing 52 2.9 Plasmid vectors 53 2.9.1 Plasmid replication 54 2.9.2 Cloning sites 55 2.9.3 Selectable markers 57 2.9.4 Insertional inactivation 58 2.9.5 Transformation 59 2.10 Vectors based on the lambda bacteriophage 61 2.10.1 Lambda biology 61 2.10.2 In vitro packaging 65 2.10.3 Insertion vectors 66 2.10.4 Replacement vectors 68 2.11 Cosmids 71 2.12 Supervectors: YACs and BACs 72 2.13 Summary 73 3 Genomic and cDNA Libraries 75 3.1 Genomic libraries 77 3.1.1 Partial digests 77 3.1.2 Choice of vectors 80 3.1.3 Construction and evaluation of a genomic library 83 3.2 Growing and storing libraries 86 3.3 cDNA libraries 87 3.3.1 Isolation of mRNA 88 3.3.2 cDNA synthesis 89 3.3.3 Bacterial cDNA 93 3.4 Screening libraries with gene probes 94 3.4.1 Hybridization 94 3.4.2 Labelling probes 98 3.4.3 Steps in a hybridization experiment 99 3.4.4 Screening procedure 100 3.4.5 Probe selection and generation 101 3.5 Screening expression libraries with antibodies 103 3.6 Characterization of plasmid clones 106 3.6.1 Southern blots 107 3.6.2 PCR and sequence analysis 108 4 Polymerase Chain Reaction (PCR) 109 4.1 The PCR reaction 110 4.2 PCR in practice 114 4.2.1 Optimisation of the PCR reaction 114 4.2.2 Primer design 115 4.2.3 Analysis of PCR products 117 4.2.4 Contamination 118 4.3 Cloning PCR products 119 4.4 Long-range PCR 121 4.5 Reverse-transcription PCR 123 4.6 Quantitative and real-time PCR 123 4.6.1 SYBR Green 123 4.6.2 TaqMan 125 4.6.3 Molecular beacons 125 4.7 Applications of PCR 127 4.7.1 Probes and other modified products 127 4.7.2 PCR cloning strategies 128 4.7.3 Analysis of recombinant clones and rare events 129 4.7.4 Diagnostic applications 130 5 Sequencing a Cloned Gene 131 5.1 DNA sequencing 131 5.1.1 Principles of DNA sequencing 131 5.1.2 Automated sequencing 136 5.1.3 Extending the sequence 137 5.1.4 Shotgun sequencing; contig assembly 138 5.2 Databank entries and annotation 140 5.3 Sequence analysis 146 5.3.1 Identification of coding region 146 5.3.2 Expression signals 147 5.4 Sequence comparisons 148 5.4.1 DNA sequences 148 5.4.2 Protein sequence comparisons 151 5.4.3 Sequence alignments: Clustal 157 5.5 Protein structure 160 5.5.1 Structure predictions 160 5.5.2 Protein motifs and domains 162 5.6 Confirming gene function 165 5.6.1 Allelic replacement and gene knockouts 166 5.6.2 Complementation 168 6 Analysis of Gene Expression 169 6.1 Analysing transcription 169 6.1.1 Northern blots 170 6.1.2 Reverse transcription-PCR 171 6.1.3 In situ hybridization 174 6.2 Methods for studying the promoter 174 6.2.1 Locating the promoter 175 6.2.2 Reporter genes 177 6.3 Regulatory elements and DNA-binding proteins 179 6.3.1 Yeast one-hybrid assays 179 6.3.2 DNase I footprinting 181 6.3.3 Gel retardation assays 181 6.3.4 Chromatin immunoprecipitation (ChIP)