Chapter 1: Introduction

1.1 The beginning of gene cloning
1.2 How to use this book
1.3 What you need to know before you read this book
1.4 A request from the authors
Further reading

Chapter 2: Genome Organisation

2.1 Introduction
2.2 The C-value paradox
2.3 The human genome
2.4 Genomes of other eukaryotes
2.5 Bacterial genomes
2.6 Plasmids
2.7 Viral genomes
2.8 GC content
2.9 Physical characteristics of eukaryotic chromosomes
2.10 Karyotype
2.11 Euchromatin and heterochromatin
2.12 CpG islands
Questions and answers
Further reading

Chapter 3: Key tools for gene cloning

3.1 Introduction
3.2 Vectors
3.3 Restriction enzymes
3.4 DNA ligase
3.5 Transformation
3.6 Purification of plasmid DNA
3.7 More restriction enzymes
3.8 Alkaline phosphatase
3.9 More about vectors
3.10 Analysing cloned DNA by restriction mapping
3.11 Measuring the size of DNA fragments
3.12 The polymerase chain reaction and its use in gene cloning
3.13 How does PCR work?
3.14 Designing PCR primers
3.15 The PCR reaction
3.16 Uses for PCR products
3.17 Cloning PCR products
3.18 Real-time PCR for quantification of DNA
3.19 Advantages and limitations of PCR
Questions and answers
Further reading

Chapter 4: Gene identification and DNA libraries

4.1 The problem
4.2 Genomic library
4.3 Constructing a genomic library
4.4 How many clones?
4.5 Some DNA fragments are under-represented in genomic libraries
4.6 Using partial digests to make a genomic library
4.7 Storage of genomic libraries
4.8 Advantages and disadvantages of genomic libraries
4.9 Cloning vectors for gene libraries
4.10 Vectors derived from bacteriophage
4.11 Packing bacteriophage in vitro
4.12 Cloning with bacteriophage
4.13 Calculating the titer of your library
4.14 Cosmid libraries
4.15 Making a cosmid library
4.16 YAC and BAC vectors
4.17 cDNA libraries
4.18 Making a cDNA library
4.19 Cloning the cDNA product
4.20 Expressed sequence tags
4.21 What are the disadvantages of a cDNA library?
Questions and answers
Further reading

Chapter 5: Screening DNA libraries

5.1 The problem
5.2 Screening methods based on gene expression
5.3 Complementation
5.4 Immunological screening of expression libraries
5.5 Screening methods based on detecting a DNA sequence
5.6 Oligonucleotide probes
5.7 Cloned DNA fragments as probes
5.8 Colony and plaque hybridization
5.9 Differential screening
5.10 Using PCR to screen a library
Questions and answers
Further reading

Chapter 6: Further routes to gene identification

6.1 How do we get from phenotype to gene: a fundamental problem in gene cloning
6.2 Gene tagging: a method that both mutates and marks genes
6.3 A simple example of transposon tagging in bacteria: cloning adhesive genes from pseudomonas
6.4 Signature-tagged mutagenesis: cloning bacterial genes with ‘difficult’ phenotypes
6.5 Gene tagging in higher eukaryotes: resistance genes in plants
6.6 Positional cloning: using maps to track down genes
6.7 Identification of a linked marker
6.8 Moving from the marker towards the gene of interest
6.9 Identifying the gene of interest
6.10 Cloning of the CF gene: a case study
Questions and answers
Further reading

Chapter 7: Sequencing DNA

7.1 Introduction
7.2 Overview of sequencing
7.3 Sanger sequencing
7.4 The Sanger sequencing protocol requires a single-stranded DNA template
7.5 Modifications of the original Sanger protocol
7.6 Strategies for sequencing a DNA fragment
7.7 High-throughput sequencing protocols
7.8 The modern sequencing protocol
7.9 Genome sequencing
7.10 High-throughput pyrosequencing
7.11 The importance of DNA sequencing
Questions and answers
Further reading

Chapter 8: Bioinformatics

8.1 Introduction
8.2 What does a gene look like?
8.3 Identifying eukaryotic genes
8.4 Sequence comparisons
8.5 Pair-wise comparisons
8.6 Identity and similarity
8.7 Is the alignment significant?
8.8 What can alignments tell us about the biology of the sequences being compared?
8.9 Similarity searches
8.10 Fasta
8.11 BLAST
8.12 What can similarity searches tell us about the biology of the sequences being compared?
8.13 Multiple sequence alignments
8.14 What can multiple sequence alignments tell us about the structure and function of proteins?
8.15 Consensus patterns and sequence motifs
8.16 Investigating the three-dimensional structures of biological molecules
8.17 Using sequence alignments to create a phylogenetic tree
Questions and answers
Further reading

Chapter 9: Production of proteins from cloned genes

9.1 Why express proteins?
9.2 Requirements for protein production from cloned genes
9.3 The use of E. coli as a host organism for protein production
9.4 Some problems in obtaining high level production of proteins in E. coli
9.5 Beyond E. coli: protein expression in eukaryotic systems
9.6 A final word about protein purification
Questions and answers
Further reading

Chapter 10: Gene cloning in the functional analysis of proteins

10.1 Introduction
10.2 Analyzing the expression and role of unknown genes
10.3 Determining the cellular location of proteins
10.4 Mapping of membrane proteins
10.5 Detecting interacting proteins
10.6 Site-directed mutagenesis for detailed probing of gene and protein function
Questions and answers
Further reading

Chapter 11: The analysis of the regulation of gene expression

11.1 Introduction
11.2 Determining the transcription start of a gene
11.3 Determining the level of gene expression
11.4 Identifying the important regulatory regions
11.5 Identifying protein factors
11.6 Global studies of gene expression
Questions and answers
Further reading

Chapter 12: The production and uses of transgenic organisms

12.1 What is a transgenic organism?
12.2 Why make transgenic organisms?
12.3 How are transgenic organisms made?
12.4 Drawbacks and problems
12.5 Knockout mice and other organisms: the growth of precision in transgene targeting
12.6 Is the technology available to produce transgenic people?
Questions and answers
Further reading

Chapter 13: Forensic and Medical Applications

13.1 Introduction
13.2 Forensics
13.3 DNA profiling
13.4 Multiplex PCR
13.5 Samples for forensic analysis
13.6 Obtaining more information from DNA profiles
13.7 Other applications of DNA profiling
13.8 Medical applications
13.9 Techniques for diagnosis of inherited disorders
13.10 Whole genome amplification
13.11 Diagnosis of infectious disease
13.12 Diagnosis and management of cancer
Questions and answers
Further reading

Glossary