Gene Cloning

ISBN-10: 0748765344

ISBN-13: 9780748765348

Edition: 2006

List price: $83.00
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Description:

This volume gives a broad overview of the subject and introduces the reader to some of the more common uses of gene cloning from applications in research to practical applications in biotechnology and medicine.
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Book details

List price: $83.00
Copyright year: 2006
Publisher: Taylor & Francis Group
Publication date: 10/24/2006
Binding: Paperback
Pages: 474
Size: 6.75" wide x 9.75" long x 1.00" tall
Weight: 1.100
Language: English

Dr Julia Lodge is Assistant Professor (Lecturer) at the School of Biosciences, University of Birmingham, UK and teaches gene cloning, genetic engineering, genomics. Her main research interest is bacterial infectious diseases.   Dr Pete Lund is Associate Professor (Senior Lecturer) at the School of Biosciences, University of Birmingham, UK and teaches cellular and molecular biology, genetics, protein structure and function. His research focuses on molecular chaperones and stress responses.   Dr Steve Minchin is Associate Professor (Senior Lecturer) at the School of Biosciences, University of Birmingham, UK and teaches genetics, gene expression, genomics. His main area of research is the regulation of gene expression.

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