From Genes to Genomes - Concepts and Applications of Dna Technology

Klappentext The latest edition of this highly successful textbook introduces the key techniques and concepts involved in cloning genes and in studying their expression and variation.The new edition features:* Increased coverage of whole-genome sequencing technologies and enhanced treatment of bioinformatics.* Clear, two-colour diagrams throughout.* A dedicated website including all figures.Noted for its outstanding balance between clarity of coverage and level of detail, this book provides an excellent introduction to the fast moving world of molecular genetics. Zusammenfassung The latest edition of this highly successful textbook introduces the key techniques and concepts involved in cloning genes and in studying their expression and variation.The new edition features:* Increased coverage of whole-genome sequencing technologies and enhanced treatment of bioinformatics.* Clear, two-colour diagrams throughout.* A dedicated website including all figures.Noted for its outstanding balance between clarity of coverage and level of detail, this book provides an excellent introduction to the fast moving world of molecular genetics. Inhaltsverzeichnis Preface xiii1 From Genes to Genomes 11.1 Introduction 11.2 Basic molecular biology 41.2.1 The DNA backbone 41.2.2 The base pairs 61.2.3 RNA structure 101.2.4 Nucleic acid synthesis 111.2.5 Coiling and supercoilin 111.3 What is a gene? 131.4 Information flow: gene expression 151.4.1 Transcription 161.4.2 Translation 191.5 Gene structure and organisation 201.5.1 Operons 201.5.2 Exons and introns 211.6 Refinements of the model 222 How to Clone a Gene 252.1 What is cloning? 252.2 Overview of the procedures 262.3 Extraction and purification of nucleic acids 292.3.1 Breaking up cells and tissues 292.3.2 Alkaline denaturation 312.3.3 Column purification 312.4 Detection and quantitation of nucleic acids 322.5 Gel electrophoresis 332.5.1 Analytical gel electrophoresis 332.5.2 Preparative gel electrophoresis 362.6 Restriction endonucleases 362.6.1 Specificity 372.6.2 Sticky and blunt ends 402.7 Ligation 422.7.1 Optimising ligation conditions 442.7.2 Preventing unwanted ligation: alkaline phosphatase and double digests 462.7.3 Other ways of joining DNA fragments 482.8 Modification of restriction fragment ends 492.8.1 Linkers and adaptors 502.8.2 Homopolymer tailing 522.9 Plasmid vectors 532.9.1 Plasmid replication 542.9.2 Cloning sites 552.9.3 Selectable markers 572.9.4 Insertional inactivation 582.9.5 Transformation 592.10 Vectors based on the lambda bacteriophage 612.10.1 Lambda biology 612.10.2 In vitro packaging 652.10.3 Insertion vectors 662.10.4 Replacement vectors 682.11 Cosmids 712.12 Supervectors: YACs and BACs 722.13 Summary 733 Genomic and cDNA Libraries 753.1 Genomic libraries 773.1.1 Partial digests 773.1.2 Choice of vectors 803.1.3 Construction and evaluation of a genomic library 833.2 Growing and storing libraries 863.3 cDNA libraries 873.3.1 Isolation of mRNA 883.3.2 cDNA synthesis 893.3.3 Bacterial cDNA 933.4 Screening libraries with gene probes 943.4.1 Hybridization 943.4.2 Labelling probes 983.4.3 Steps in a hybridization experiment 993.4.4 Screening procedure 1003.4.5 Probe selection and generation 1013.5 Screening expression libraries with antibodies 1033.6 Characterization of plasmid clones 1063.6.1 Southern blots 1073.6.2 PCR and sequence analysis 1084 Polymerase Chain Reaction (PCR) 1094.1 The PCR reaction 1104.2 PCR in practice 1144.2.1 Optimisation of the PCR reaction 1144.2.2 Primer design 1154.2.3 Analysis of PCR products 1174.2.4 Contamination 1184.3 Cloning PCR products 1194.4 Long-range PCR 1214.5 Reverse-transcription PCR 1234.6 Quantitative and real-time PCR 1234.6.1 SYBR Green 1234.6.2 TaqMan 1254.6.3 Molecular beacons 1254.7 Applications of PCR 1274.7....

List of contents

Preface xiii
 
1 From Genes to Genomes 1
 
1.1 Introduction 1
 
1.2 Basic molecular biology 4
 
1.2.1 The DNA backbone 4
 
1.2.2 The base pairs 6
 
1.2.3 RNA structure 10
 
1.2.4 Nucleic acid synthesis 11
 
1.2.5 Coiling and supercoilin 11
 
1.3 What is a gene? 13
 
1.4 Information flow: gene expression 15
 
1.4.1 Transcription 16
 
1.4.2 Translation 19
 
1.5 Gene structure and organisation 20
 
1.5.1 Operons 20
 
1.5.2 Exons and introns 21
 
1.6 Refinements of the model 22
 
2 How to Clone a Gene 25
 
2.1 What is cloning? 25
 
2.2 Overview of the procedures 26
 
2.3 Extraction and purification of nucleic acids 29
 
2.3.1 Breaking up cells and tissues 29
 
2.3.2 Alkaline denaturation 31
 
2.3.3 Column purification 31
 
2.4 Detection and quantitation of nucleic acids 32
 
2.5 Gel electrophoresis 33
 
2.5.1 Analytical gel electrophoresis 33
 
2.5.2 Preparative gel electrophoresis 36
 
2.6 Restriction endonucleases 36
 
2.6.1 Specificity 37
 
2.6.2 Sticky and blunt ends 40
 
2.7 Ligation 42
 
2.7.1 Optimising ligation conditions 44
 
2.7.2 Preventing unwanted ligation: alkaline phosphatase and double digests 46
 
2.7.3 Other ways of joining DNA fragments 48
 
2.8 Modification of restriction fragment ends 49
 
2.8.1 Linkers and adaptors 50
 
2.8.2 Homopolymer tailing 52
 
2.9 Plasmid vectors 53
 
2.9.1 Plasmid replication 54
 
2.9.2 Cloning sites 55
 
2.9.3 Selectable markers 57
 
2.9.4 Insertional inactivation 58
 
2.9.5 Transformation 59
 
2.10 Vectors based on the lambda bacteriophage 61
 
2.10.1 Lambda biology 61
 
2.10.2 In vitro packaging 65
 
2.10.3 Insertion vectors 66
 
2.10.4 Replacement vectors 68
 
2.11 Cosmids 71
 
2.12 Supervectors: YACs and BACs 72
 
2.13 Summary 73
 
3 Genomic and cDNA Libraries 75
 
3.1 Genomic libraries 77
 
3.1.1 Partial digests 77
 
3.1.2 Choice of vectors 80
 
3.1.3 Construction and evaluation of a genomic library 83
 
3.2 Growing and storing libraries 86
 
3.3 cDNA libraries 87
 
3.3.1 Isolation of mRNA 88
 
3.3.2 cDNA synthesis 89
 
3.3.3 Bacterial cDNA 93
 
3.4 Screening libraries with gene probes 94
 
3.4.1 Hybridization 94
 
3.4.2 Labelling probes 98
 
3.4.3 Steps in a hybridization experiment 99
 
3.4.4 Screening procedure 100
 
3.4.5 Probe selection and generation 101
 
3.5 Screening expression libraries with antibodies 103
 
3.6 Characterization of plasmid clones 106
 
3.6.1 Southern blots 107
 
3.6.2 PCR and sequence analysis 108
 
4 Polymerase Chain Reaction (PCR) 109
 
4.1 The PCR reaction 110
 
4.2 PCR in practice 114
 
4.2.1 Optimisation of the PCR reaction 114
 
4.2.2 Primer design 115
 
4.2.3 Analysis of PCR products 117
 
4.2.4 Contamination 118
 
4.3 Cloning PCR products 119
 
4.4 Long-range PCR 121
 
4.5 Reverse-transcription PCR 123
 
4.6 Quantitative and real-time PCR 123
 
4.6.1 SYBR Green 123
 
4.6.2 TaqMan 125
 
4.6.3 Molecular beacons 125
 
4.7 Applications of PCR 127
 
4.7.1 Probes and other modified products 127
 
4.7.2 PCR cloning strategies 128
 
4.7.3 Analysis of recombinant clones and rare events 129

Report

"This third edition is absolutely necessary to incorporate the recent advances, such as genome sequencing, polymerase chain reaction, and microarray technology, in this field." ( Doody's , 19 October 2012)

Product details

• Authors: Dale, Jeremy Dale, Jeremy W Dale, Jeremy W. Dale, Jeremy W. (University of Surrey Dale, Jeremy W. Schantz Dale, Jeremy W. Von Schantz Dale, Jeremy W./ Von Schantz Dale, Jw Dale, DALE JEREMY W SCHANTZ MALCOLM V, Dale Jeremy W., Nichola Plant, Nicholas Plant, Malcolm vo Schantz, Malcolm Von Schantz, Malcolm von Schantz
• Publisher: Wiley, John and Sons Ltd
• Languages: English
• Product format: Hardback
• Released: 01.12.2011
• EAN: 9780470683866
• ISBN: 978-0-470-68386-6
• No. of pages: 400
• Subjects: Natural sciences, medicine, IT, technology > Biology > Genetics, genetic engineering; Molekulargenetik, Life Sciences, Biowissenschaften, Molecular Genetics, Genklonierung, Gene Cloning