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| Introduction | |
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| The Beginning of Gene Cloning | |
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| How To Use This Book | |
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| What You Need To Know Before You Read This Book | |
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| A Request From the Authors | |
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| Further Reading | |
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| Genome Organization | |
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| Introduction | |
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| The C-value Paradox | |
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| The Human Genome | |
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| Genomes of Other Eukaryotes | |
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| Bacterial Genomes | |
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| Plasmids | |
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| Viral Genomes | |
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| GC Content | |
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| Physical Characteristics of Eukaryotic Chromosomes | |
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| Karyotype | |
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| Euchromatin and Heterochromatin | |
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| CpG Islands | |
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| Questions and Answers | |
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| Further Reading | |
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| Key Tools for Gene Cloning | |
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| Introduction | |
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| Vectors | |
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| Restriction Enzymes | |
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| DNA Ligase | |
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| Transformation | |
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| Purification of Plasmid DNA | |
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| More Restriction Enzymes | |
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| Alkaline Phosphatase | |
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| More About Vectors | |
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| Analyzing Cloned DNA by Restriction Mapping | |
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| Measuring the Size of DNA Fragments | |
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| The Polymerase Chain Reaction and Its Use in Gene Cloning | |
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| How Does PCR Work? | |
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| Designing PCR Primers | |
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| The PCR Reaction | |
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| Uses for PCR Products | |
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| Cloning PCR Products | |
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| Real-time PCR for Quantification of DNA | |
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| Advantages and Limitations of PCR | |
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| Questions and Answers | |
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| Further Reading | |
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| Gene Identification and DNA Libraries | |
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| The Problem | |
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| Genomic Library | |
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| Constructing a Genomic Library | |
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| How Many Clones? | |
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| Some DNA Fragments are Under-represented in Genomic Libraries | |
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| Using Partial Digests to Make a Genomic Library | |
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| Storage of Genomic Libraries | |
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| Advantages and Disadvantages of Genomic Libraries | |
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| Cloning Vectors for Gene Libraries | |
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| Vectors Derived from Bacteriophage [lambda] | |
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| Packing Bacteriophage [lambda] In Vitro | |
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| Cloning with Bacteriophage [lambda] | |
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| Calculating the Titer of your Library | |
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| Cosmid Libraries | |
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| Making a Cosmid Library | |
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| YAC and BAC Vectors | |
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| cDNA Libraries | |
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| Making a cDNA Library | |
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| Cloning the cDNA Product | |
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| Expressed Sequence Tags | |
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| What are the Disadvantages of a cDNA Library? | |
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| Questions and Answers | |
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| Further Reading | |
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| Screening DNA Libraries | |
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| The Problem | |
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| Screening Methods Based on Gene Expression | |
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| Complementation | |
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| Immunological Screening of Expression Libraries | |
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| Screening Methods Based on Detecting a DNA Sequence | |
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| Oligonucleotide Probes | |
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| Cloned DNA Fragments as Probes | |
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| Colony and Plaque Hybridization | |
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| Differential Screening | |
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| Using PCR to Screen a Library | |
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| Questions and Answers | |
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| Further Reading | |
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| Further Routes to Gene Identification | |
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| How Do We Get From Phenotype to Gene: a Fundamental Problem in Gene Cloning | |
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| Gene Tagging: A Method That Both Mutates and Marks Genes | |
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| A Simple Example of Transposon Tagging in Bacteria: Cloning Adherence Genes from Pseudomonas | |
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| Signature-tagged Mutagenesis: Cloning Bacterial Genes with "Difficult" Phenotypes | |
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| Gene Tagging in Higher Eukaryotes: Resistance Genes in Plants | |
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| Positional Cloning: Using Maps to Track Down Genes | |
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| Identification of a Linked Marker | |
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| Moving From the Marker Towards the Gene of Interest | |
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| Identifying the Gene of Interest | |
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| Cloning of the CF Gene: A Case Study | |
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| Questions and Answers | |
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| Further Reading | |
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| Sequencing DNA | |
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| Introduction | |
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| Overview of Sequencing | |
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| Sanger Sequencing | |
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| The Sanger Sequencing Protocol Requires a Single-stranded DNA Template | |
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| Modifications of the Original Sanger Protocol | |
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| Strategies for Sequencing a DNA Fragment | |
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| High-throughput Sequencing Protocols | |
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| The Modern Sequencing Protocol | |
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| Genome Sequencing | |
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| High-throughput Pyrosequencing | |
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| The Importance of DNA Sequencing | |
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| Questions and Answers | |
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| Further Reading | |
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| Bioinformatics | |
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| Introduction | |
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| What Does a Gene Look Like? | |
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| Identifying Eukaryotic Genes | |
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| Sequence Comparisons | |
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| Pair-wise Comparisons | |
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| Identity and Similarity | |
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| Is the Alignment Significant? | |
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| What Can Alignments Tell Us About the Biology of the Sequences Being Compared? | |
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| Similarity Searches | |
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| Fasta | |
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| BLAST | |
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| What Can Similarity Searches Tell Us About the Biology of the Sequences Being Compared? | |
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| Multiple Sequence Alignments | |
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| What Can Multiple Sequence Alignments Tell Us About the Structure and Function of Proteins? | |
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| Consensus Patterns and Sequence Motifs | |
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| Investigating the Three-dimensional Structures of Biological Molecules | |
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| Using Sequence Alignments to Create a Phylogenetic Tree | |
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| Questions and Answers | |
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| Further Reading | |
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| Production of Proteins from Cloned Genes | |
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| Why Express Proteins? | |
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| Requirements for Protein Production from Cloned Genes | |
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| The Use of E. coli as a Host Organism for Protein Production | |
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| Some Problems in Obtaining High Level Production of Proteins in E. coli | |
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| Beyond E. coli: Protein Expression in Eukaryotic Systems | |
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| A Final Word About Protein Purification | |
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| Questions and Answers | |
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| Further Reading | |
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| Gene Cloning in the Functional Analysis of Proteins | |
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| Introduction | |
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| Analyzing the Expression and Role of Unknown Genes | |
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| Determining the Cellular Location of Proteins | |
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| Mapping of Membrane Proteins | |
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| Detecting Interacting Proteins | |
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| Site-Directed Mutagenesis for Detailed Probing of Gene and Protein Function | |
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| Questions and Answers | |
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| Further Reading | |
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| The Analysis of the Regulation of Gene Expression | |
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| Introduction | |
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| Determining the Transcription Start of a Gene | |
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| Determining the Level of Gene Expression | |
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| Identifying the Important Regulatory Regions | |
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| Identifying Protein Factors | |
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| Global Studies of Gene Expression | |
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| Questions and Answers | |
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| Further Reading | |
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| The Production and Uses of Transgenic Organisms | |
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| What is a Transgenic Organism? | |
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| Why Make Transgenic Organisms? | |
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| How are Transgenic Organisms Made? | |
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| Drawbacks and Problems | |
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| Knockout Mice and Other Organisms: The Growth of Precision in Transgene Targeting | |
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| Is the Technology Available to Produce Transgenic People? | |
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| Questions and Answers | |
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| Further Reading | |
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| Forensic and Medical Applications | |
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| Introduction | |
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| Forensics | |
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| DNA Profiling | |
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| Multiplex PCR | |
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| Samples for Forensic Analysis | |
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| Obtaining More Information from DNA Profiles | |
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| Other Applications of DNA Profiling | |
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| Medical Applications | |
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| Techniques for Diagnosis of Inherited Disorders | |
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| Whole Genome Amplification | |
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| Diagnosis of Infectious Disease | |
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| Diagnosis and Management of Cancer | |
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| Questions and Answers | |
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| Further Reading | |
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| Glossary | |
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| Index | |