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Gene Cloning - Julia Lodge

Paperback

Published: 1st September 2006
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The ability to successfully clone genes underlies the majority of our knowledge in molecular and cellular biology. Gene Cloning introduces the diverse array of techniques available to clone genes and how they can be used effectively both in the research laboratory, to gain knowledge about the gene, and for use in biotechnology, medicine, the pharmaceutical industry, and agriculture. It shows how cloning genes is an integral part of genomics and underlines its relevance in the post-genomic age, as a tool required to test predictions of gene regulation and function made through bioinformatics. Applications of gene cloning in medicine, both for diagnosis and treatment, and in the pharmaceutical industry and agriculture, are also covered in the book.

Gene Cloning takes a fresh approach to teaching molecular and cellular biology and will be a valuable resource to both undergraduates and lecturers of biological and biomedical science courses.

'Had I ever been a practical molecular biologist, this is the book that would reassure me of the principles behind my experiments. Furthermore, if, for example, I wanted to find the transcription starting point of a gene, this would tell me the theory behind how to do it. Thus it is not a lab cookbook or manual, but a very user-friendly and helpful adviser. I was rather impressed with this book and will suggest it as a text, not only for my second and third year students but also for our MSc molecular genetics course.' - The Times Higher Education Supplement, February 23rd 2007 'Instructors seeking a text for a course in molecular biology would be wise to consider this one. The authors are to be commended for a readable and well organized presentation. This is a strong effort. The presentation is logical, the level correct. Were I to teach a course in this area, this book would be high on my list.' - Eugene A Davidson, PhD, Georgetown University School of Medicine, Doody's Reviews, April 2007 'The writing is clear and concise and, importantly for beginners, the essential information is not lost amongst a myriad of details. The instructor will find an excellent combination of traditional and up-to-date topics treated and will be able to easily complement this with information from the literature or laboratory.' - BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION, Vol. 36, 2008

Introduction
The Beginning of Gene Cloningp. 1
How To Use This Bookp. 3
What You Need To Know Before You Read This Bookp. 5
A Request From the Authorsp. 5
Further Readingp. 6
Genome Organization
Introductionp. 7
The C-value Paradoxp. 8
The Human Genomep. 9
Genomes of Other Eukaryotesp. 19
Bacterial Genomesp. 24
Plasmidsp. 25
Viral Genomesp. 26
GC Contentp. 27
Physical Characteristics of Eukaryotic Chromosomesp. 28
Karyotypep. 28
Euchromatin and Heterochromatinp. 30
CpG Islandsp. 31
Questions and Answersp. 32
Further Readingp. 34
Key Tools for Gene Cloning
Introductionp. 35
Vectorsp. 36
Restriction Enzymesp. 38
DNA Ligasep. 40
Transformationp. 42
Purification of Plasmid DNAp. 45
More Restriction Enzymesp. 47
Alkaline Phosphatasep. 51
More About Vectorsp. 53
Analyzing Cloned DNA by Restriction Mappingp. 58
Measuring the Size of DNA Fragmentsp. 59
The Polymerase Chain Reaction and Its Use in Gene Cloningp. 64
How Does PCR Work?p. 67
Designing PCR Primersp. 72
The PCR Reactionp. 73
Uses for PCR Productsp. 74
Cloning PCR Productsp. 74
Real-time PCR for Quantification of DNAp. 76
Advantages and Limitations of PCRp. 76
Questions and Answersp. 78
Further Readingp. 83
Gene Identification and DNA Libraries
The Problemp. 85
Genomic Libraryp. 87
Constructing a Genomic Libraryp. 87
How Many Clones?p. 89
Some DNA Fragments are Under-represented in Genomic Librariesp. 90
Using Partial Digests to Make a Genomic Libraryp. 90
Storage of Genomic Librariesp. 92
Advantages and Disadvantages of Genomic Librariesp. 92
Cloning Vectors for Gene Librariesp. 93
Vectors Derived from Bacteriophage [lambda]p. 93
Packing Bacteriophage [lambda] In Vitrop. 95
Cloning with Bacteriophage [lambda]p. 97
Calculating the Titer of your Libraryp. 98
Cosmid Librariesp. 98
Making a Cosmid Libraryp. 99
YAC and BAC Vectorsp. 100
cDNA Librariesp. 101
Making a cDNA Libraryp. 103
Cloning the cDNA Productp. 105
Expressed Sequence Tagsp. 108
What are the Disadvantages of a cDNA Library?p. 108
Questions and Answersp. 109
Further Readingp. 116
Screening DNA Libraries
The Problemp. 117
Screening Methods Based on Gene Expressionp. 118
Complementationp. 119
Immunological Screening of Expression Librariesp. 120
Screening Methods Based on Detecting a DNA Sequencep. 123
Oligonucleotide Probesp. 124
Cloned DNA Fragments as Probesp. 127
Colony and Plaque Hybridizationp. 127
Differential Screeningp. 132
Using PCR to Screen a Libraryp. 133
Questions and Answersp. 135
Further Readingp. 140
Further Routes to Gene Identification
How Do We Get From Phenotype to Gene: a Fundamental Problem in Gene Cloningp. 141
Gene Tagging: A Method That Both Mutates and Marks Genesp. 142
A Simple Example of Transposon Tagging in Bacteria: Cloning Adherence Genes from Pseudomonasp. 149
Signature-tagged Mutagenesis: Cloning Bacterial Genes with "Difficult" Phenotypesp. 152
Gene Tagging in Higher Eukaryotes: Resistance Genes in Plantsp. 156
Positional Cloning: Using Maps to Track Down Genesp. 159
Identification of a Linked Markerp. 161
Moving From the Marker Towards the Gene of Interestp. 161
Identifying the Gene of Interestp. 166
Cloning of the CF Gene: A Case Studyp. 168
Questions and Answersp. 169
Further Readingp. 171
Sequencing DNA
Introductionp. 173
Overview of Sequencingp. 174
Sanger Sequencingp. 175
The Sanger Sequencing Protocol Requires a Single-stranded DNA Templatep. 179
Modifications of the Original Sanger Protocolp. 181
Strategies for Sequencing a DNA Fragmentp. 182
High-throughput Sequencing Protocolsp. 184
The Modern Sequencing Protocolp. 185
Genome Sequencingp. 188
High-throughput Pyrosequencingp. 197
The Importance of DNA Sequencingp. 202
Questions and Answersp. 203
Further Readingp. 205
Bioinformatics
Introductionp. 207
What Does a Gene Look Like?p. 208
Identifying Eukaryotic Genesp. 214
Sequence Comparisonsp. 217
Pair-wise Comparisonsp. 217
Identity and Similarityp. 220
Is the Alignment Significant?p. 222
What Can Alignments Tell Us About the Biology of the Sequences Being Compared?p. 224
Similarity Searchesp. 224
Fastap. 226
BLASTp. 228
What Can Similarity Searches Tell Us About the Biology of the Sequences Being Compared?p. 230
Multiple Sequence Alignmentsp. 233
What Can Multiple Sequence Alignments Tell Us About the Structure and Function of Proteins?p. 234
Consensus Patterns and Sequence Motifsp. 235
Investigating the Three-dimensional Structures of Biological Moleculesp. 237
Using Sequence Alignments to Create a Phylogenetic Treep. 239
Questions and Answersp. 242
Further Readingp. 246
Production of Proteins from Cloned Genes
Why Express Proteins?p. 249
Requirements for Protein Production from Cloned Genesp. 252
The Use of E. coli as a Host Organism for Protein Productionp. 252
Some Problems in Obtaining High Level Production of Proteins in E. colip. 260
Beyond E. coli: Protein Expression in Eukaryotic Systemsp. 265
A Final Word About Protein Purificationp. 274
Questions and Answersp. 275
Further Readingp. 277
Gene Cloning in the Functional Analysis of Proteins
Introductionp. 279
Analyzing the Expression and Role of Unknown Genesp. 280
Determining the Cellular Location of Proteinsp. 290
Mapping of Membrane Proteinsp. 293
Detecting Interacting Proteinsp. 297
Site-Directed Mutagenesis for Detailed Probing of Gene and Protein Functionp. 304
Questions and Answersp. 309
Further Readingp. 312
The Analysis of the Regulation of Gene Expression
Introductionp. 315
Determining the Transcription Start of a Genep. 318
Determining the Level of Gene Expressionp. 326
Identifying the Important Regulatory Regionsp. 338
Identifying Protein Factorsp. 350
Global Studies of Gene Expressionp. 353
Questions and Answersp. 361
Further Readingp. 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 Problemsp. 396
Knockout Mice and Other Organisms: The Growth of Precision in Transgene Targetingp. 398
Is the Technology Available to Produce Transgenic People?p. 406
Questions and Answersp. 407
Further Readingp. 410
Forensic and Medical Applications
Introductionp. 411
Forensicsp. 411
DNA Profilingp. 413
Multiplex PCRp. 414
Samples for Forensic Analysisp. 415
Obtaining More Information from DNA Profilesp. 416
Other Applications of DNA Profilingp. 417
Medical Applicationsp. 418
Techniques for Diagnosis of Inherited Disordersp. 422
Whole Genome Amplificationp. 435
Diagnosis of Infectious Diseasep. 437
Diagnosis and Management of Cancerp. 439
Questions and Answersp. 441
Further Readingp. 444
Glossaryp. 445
Indexp. 453
Table of Contents provided by Ingram. All Rights Reserved.

ISBN: 9780748765348
ISBN-10: 0748765344
Audience: Tertiary; University or College
Format: Paperback
Language: English
Number Of Pages: 462
Published: 1st September 2006
Country of Publication: GB
Dimensions (cm): 24.13 x 17.27  x 2.29
Weight (kg): 0.91
Edition Number: 1