Gene Cloning

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ISBN: 9780748765348 | 0748765344
Cover: Nonspecific Binding
Copyright: 10/24/2006

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Cloning genes has become such a routine part of molecular biology, and the approaches to it so many and diverse, undergraduates can easily be overwhelmed. Gene Cloning is designed to give the undergraduate a broad overview of the subject, guiding the student through the basic elements involved and their utility, and further through the interpretation of results and datasets. Having understood the basic processes, the reader is introduced to some of the most common uses of gene cloning, from applications with more research-oriented goals to practical applications in biotechnology in medicine. Clearly presented in two-colour text, Gene Cloning is essential reading for undergraduates in the molecular life sciences.

Introduction
The Beginning of Gene Cloning	p. 1
How To Use This Book	p. 3
What You Need To Know Before You Read This Book	p. 5
A Request From the Authors	p. 5
Further Reading	p. 6
Genome Organization
Introduction	p. 7
The C-value Paradox	p. 8
The Human Genome	p. 9
Genomes of Other Eukaryotes	p. 19
Bacterial Genomes	p. 24
Plasmids	p. 25
Viral Genomes	p. 26
GC Content	p. 27
Physical Characteristics of Eukaryotic Chromosomes	p. 28
Karyotype	p. 28
Euchromatin and Heterochromatin	p. 30
CpG Islands	p. 31
Questions and Answers	p. 32
Further Reading	p. 34
Key Tools for Gene Cloning
Introduction	p. 35
Vectors	p. 36
Restriction Enzymes	p. 38
DNA Ligase	p. 40
Transformation	p. 42
Purification of Plasmid DNA	p. 45
More Restriction Enzymes	p. 47
Alkaline Phosphatase	p. 51
More About Vectors	p. 53
Analyzing Cloned DNA by Restriction Mapping	p. 58
Measuring the Size of DNA Fragments	p. 59
The Polymerase Chain Reaction and Its Use in Gene Cloning	p. 64
How Does PCR Work?	p. 67
Designing PCR Primers	p. 72
The PCR Reaction	p. 73
Uses for PCR Products	p. 74
Cloning PCR Products	p. 74
Real-time PCR for Quantification of DNA	p. 76
Advantages and Limitations of PCR	p. 76
Questions and Answers	p. 78
Further Reading	p. 83
Gene Identification and DNA Libraries
The Problem	p. 85
Genomic Library	p. 87
Constructing a Genomic Library	p. 87
How Many Clones?	p. 89
Some DNA Fragments are Under-represented in Genomic Libraries	p. 90
Using Partial Digests to Make a Genomic Library	p. 90
Storage of Genomic Libraries	p. 92
Advantages and Disadvantages of Genomic Libraries	p. 92
Cloning Vectors for Gene Libraries	p. 93
Vectors Derived from Bacteriophage [lambda]	p. 93
Packing Bacteriophage [lambda] In Vitro	p. 95
Cloning with Bacteriophage [lambda]	p. 97
Calculating the Titer of your Library	p. 98
Cosmid Libraries	p. 98
Making a Cosmid Library	p. 99
YAC and BAC Vectors	p. 100
cDNA Libraries	p. 101
Making a cDNA Library	p. 103
Cloning the cDNA Product	p. 105
Expressed Sequence Tags	p. 108
What are the Disadvantages of a cDNA Library?	p. 108
Questions and Answers	p. 109
Further Reading	p. 116
Screening DNA Libraries
The Problem	p. 117
Screening Methods Based on Gene Expression	p. 118
Complementation	p. 119
Immunological Screening of Expression Libraries	p. 120
Screening Methods Based on Detecting a DNA Sequence	p. 123
Oligonucleotide Probes	p. 124
Cloned DNA Fragments as Probes	p. 127
Colony and Plaque Hybridization	p. 127
Differential Screening	p. 132
Using PCR to Screen a Library	p. 133
Questions and Answers	p. 135
Further Reading	p. 140
Further Routes to Gene Identification
How Do We Get From Phenotype to Gene: a Fundamental Problem in Gene Cloning	p. 141
Gene Tagging: A Method That Both Mutates and Marks Genes	p. 142
A Simple Example of Transposon Tagging in Bacteria: Cloning Adherence Genes from Pseudomonas	p. 149
Signature-tagged Mutagenesis: Cloning Bacterial Genes with "Difficult" Phenotypes	p. 152
Gene Tagging in Higher Eukaryotes: Resistance Genes in Plants	p. 156
Positional Cloning: Using Maps to Track Down Genes	p. 159
Identification of a Linked Marker	p. 161
Moving From the Marker Towards the Gene of Interest	p. 161
Identifying the Gene of Interest	p. 166
Cloning of the CF Gene: A Case Study	p. 168
Questions and Answers	p. 169
Further Reading	p. 171
Sequencing DNA
Introduction	p. 173
Overview of Sequencing	p. 174
Sanger Sequencing	p. 175
The Sanger Sequencing Protocol Requires a Single-stranded DNA Template	p. 179
Modifications of the Original Sanger Protocol	p. 181
Strategies for Sequencing a DNA Fragment	p. 182
High-throughput Sequencing Protocols	p. 184
The Modern Sequencing Protocol	p. 185
Genome Sequencing	p. 188
High-throughput Pyrosequencing	p. 197
The Importance of DNA Sequencing	p. 202
Questions and Answers	p. 203
Further Reading	p. 205
Bioinformatics
Introduction	p. 207
What Does a Gene Look Like?	p. 208
Identifying Eukaryotic Genes	p. 214
Sequence Comparisons	p. 217
Pair-wise Comparisons	p. 217
Identity and Similarity	p. 220
Is the Alignment Significant?	p. 222
What Can Alignments Tell Us About the Biology of the Sequences Being Compared?	p. 224
Similarity Searches	p. 224
Fasta	p. 226
BLAST	p. 228
What Can Similarity Searches Tell Us About the Biology of the Sequences Being Compared?	p. 230
Multiple Sequence Alignments	p. 233
What Can Multiple Sequence Alignments Tell Us About the Structure and Function of Proteins?	p. 234
Consensus Patterns and Sequence Motifs	p. 235
Investigating the Three-dimensional Structures of Biological Molecules	p. 237
Using Sequence Alignments to Create a Phylogenetic Tree	p. 239
Questions and Answers	p. 242
Further Reading	p. 246
Production of Proteins from Cloned Genes
Why Express Proteins?	p. 249
Requirements for Protein Production from Cloned Genes	p. 252
The Use of E. coli as a Host Organism for Protein Production	p. 252
Some Problems in Obtaining High Level Production of Proteins in E. coli	p. 260
Beyond E. coli: Protein Expression in Eukaryotic Systems	p. 265
A Final Word About Protein Purification	p. 274
Questions and Answers	p. 275
Further Reading	p. 277
Gene Cloning in the Functional Analysis of Proteins
Introduction	p. 279
Analyzing the Expression and Role of Unknown Genes	p. 280
Determining the Cellular Location of Proteins	p. 290
Mapping of Membrane Proteins	p. 293
Detecting Interacting Proteins	p. 297
Site-Directed Mutagenesis for Detailed Probing of Gene and Protein Function	p. 304
Questions and Answers	p. 309
Further Reading	p. 312
The Analysis of the Regulation of Gene Expression
Introduction	p. 315
Determining the Transcription Start of a Gene	p. 318
Determining the Level of Gene Expression	p. 326
Identifying the Important Regulatory Regions	p. 338
Identifying Protein Factors	p. 350
Global Studies of Gene Expression	p. 353
Questions and Answers	p. 361
Further Reading	p. 364
The Production and Uses of Transgenic Organisms
What is a Transgenic Organism?	p. 365
Why Make Transgenic Organisms?	p. 367
How are Transgenic Organisms Made?	p. 377
Drawbacks and Problems	p. 396
Knockout Mice and Other Organisms: The Growth of Precision in Transgene Targeting	p. 398
Is the Technology Available to Produce Transgenic People?	p. 406
Questions and Answers	p. 407
Further Reading	p. 410
Forensic and Medical Applications
Introduction	p. 411
Forensics	p. 411
DNA Profiling	p. 413
Multiplex PCR	p. 414
Samples for Forensic Analysis	p. 415
Obtaining More Information from DNA Profiles	p. 416
Other Applications of DNA Profiling	p. 417
Medical Applications	p. 418
Techniques for Diagnosis of Inherited Disorders	p. 422
Whole Genome Amplification	p. 435
Diagnosis of Infectious Disease	p. 437
Diagnosis and Management of Cancer	p. 439
Questions and Answers	p. 441
Further Reading	p. 444
Glossary	p. 445
Index	p. 453
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