Preface XI
Acknowledgement XIII
About the cover XV
1 Introduction 1
1.1 General Background 1
1.2 Short Historical Review of the Chromatography of
Nucleic Acids 6
1.3 Terms and Definitions 8
1.4 Scope and Organization of This Book 10
References 11
2 Instrumentation and Operation 14
2.1 Introduction 14
2.2 General Description of the DNA Chromatograph 15
2.3 Detailed Description of the DNA Chromatograph 16
2.3.1 The General Instrument and Materials 162.3.2 Dead Volume 17
2.3.3 Degassing the Eluent 18
2.3.4 Pumps 19
2.3.5 Gradient Formation 21
2.3.6 Pressure 23
2.3.7 Autosampler Injector 23
2.3.8 Separation Column 25
2.3.9 Column Protection 26
2.3.10 Column Oven 27
2.3.11 Detection 28
2.3.11.1 Selective vs. General Detection 28
2.3.11.2 Ultraviolet-Visible Detectors 29
2.3.11.3 Fluorescence Detector 31
2.3.11.4 Mass Spectrometry Detection 34
2.3.12 Data Analysis 37
2.3.12.1 Size Analysis 38
2.3.12.2 Peak Shape or Pattern of Peaks 38
2.3.12.3 Quantification 39
2.3.13 Fragment Collection 40
References 41
3 Chromatographic Principles for DNA Separation 42
3.1 Introduction 42
3.2 Comparison of Chromatography and Gel Electrophoresis 45
3.3 Basic Chromatographic Considerations 48
3.3.1 Retention 49
3.3.2 Retention Factors 50
3.3.3 Peak Width 50
3.3.4 Plate Theory of Chromatography 51
3.3.5 The Rate Theory of Chromatography 55
3.3.5.1 General Considerations 55
3.3.5.2 Extra Column Effects 57
3.4 Reverse Phase Column Packing Materials 57
3.4.1 Types of Materials 57
3.4.2 Polymeric Resins 59
3.4.2.1 Substrate and Crosslinking 59
3.4.2.2 Porous and Nonporous Resins 59
3.4.2.3 Monolith Polymeric Columns 62
3.4.2.4 Functionalization of the Polymer 62
3.4.3 Silica-based Materials 64
3.4.3.1 General Description 64
3.4.3.2 Functionalization 65
3.4 Reverse Phase Column Packing Materials 57
3.4.1 Types of Materials 57
3.4.2 Polymeric Resins 59
3.4.2.1 Substrate and Crosslinking 59
3.4.2.2 Porous and Nonporous Resins 59
3.4.2.3 Monolith Polymeric Columns 62
3.4.2.4 Functionalization of the Polymer 62
3.4.3 Silica-based Materials 64
3.4.3.1 General Description 64
3.4.3.2 Functionalization 65
3.5 Reverse Phase Ion Pairing Chromatography 66
3.5.1 Principles 66
3.5.2 Temperature Modes of DNA Chromatography 71
3.5.3 Effect of Metal Contamination 72
3.6 Ion Exchange Materials and Separation Mechanism 75
3.6.1 Polymer-based Anion Exchangers (Anex) 75
3.6.2 Silica-based Anion Exchangers 77
3.6.3 Basis for Separation 78
References 79
4 DHPLC 81
4.1 Introduction 81
4.2 Practice of the Technique 83
4.2.1 Melting Phenomena and Domains 83
4.2.2 Temperature Prediction 85
4.2.3 Primer Optimization and Clamping 88
4.2.4 PCR Fidelity 91
4.2.5 High-sensitivity DHPLC Determinations 92
4.2.5.1 Chromatographic Resolution between Heteroduplices
and Homoduplices 93
4.2.5.2 Mass Sensitivity for the Resolved Heteroduplices 93
4.2.5.3 PCR-induced Background 95
4.2.5.4 Example Application: Detection of Varying Levels of k-ras Alleles 96
4.3 Review of DHPLC Publications 98
4.4 Conclusions 103
References 105
5 Size Based Separations 108
5.1 Introduction 108
5.2 Fundamental Developments 108
5.3 Calibration 110
5.4 Applications 114
5.4.1 Primer extension and near-size-based separations 114
5.4.2 LOH and other size-based genotyping techniques 115
5.4.3 Size Based Purification Procedures 115
References 116
6 Purification of Nucleic Acids 118
6.1 Introduction 118
6.2 System Dead Volume 119
6.3 Cleaning 120
6.4 Testing the Instrument Operation 122
6.5 Calibration and Separation Conditions 122
6.5.1 Internal and External Calibration 122
6.5.2 Isocratic Elution 123
6.6 Software Collection Methods 132
6.7 Recovery of Material 133
References 134
7 RNA Chromatography 135
7.1 Introduction 135
7.2 Biological Extraction of RNA 137
7.3 Size Based RNA Separation 139
7.4 Separation of Cellular RNA Species 141
7.4.1 Separation of Messenger RNA from Ribosomal RNA 141
7.4.2 Analysis of Transfer RNA 143
7.5 Chromatography and Analysis of Synthetic Oligoribonucleotides 145
7.6 Application of RNA and DNA Chromatography in
cDNA Library Synthesis 149
7.7 Analysis of Gene Expression by RNA and DNA Chromatography 152
7.7.1 DNA Chromatography Analyses of RT-PCR and
Competitive RT-PCR Products 152
7.7.2 Alternative Splicing 154
7.7.3 Differential Messenger RNA Display via DNA Chromatography 155
References 158
8 Special Techniques 160
8.1 Introduction 160
8.2 Analytical and Preparative Enzymatic Cleavage of DNA 161
8.3 Analysis of DNA Methylation 164
8.4 Nucleic Acid Enzymology 171
8.4.1 Telomerase Assays 171
8.4.2 Polynucleotide Kinase Assays 173
8.4.3 Uracil DNA Glycosylase Assays 174
8.5 Protein Nucleic Acid Interaction Mapping: aFootprintingo 175
Method Section 177
8.6 Nucleic Acid Tagging 180
8.7 DNA Chromatography with Intercalating Dyes 181
References 182
9 Looking Forward 184
Appendix 1 Glossary of Terms 187
Appendix 2 System Cleaning and Passivation Treatment 207
A2.1 Background Information 207
A2.2 Reagents 211
A2.3 Preparation of the System 211
A2.4 Passivation of System 211
A2.5 Equilibration of System 212
A2.6 Passivation of Injection Port and Injection Needle 212
References 213
Appendix 3 Frequently Asked DHPLC Questions 214
A3.1 What are the various methods for DHPLC temperature selection? 214
A3.2 Does having the optimum oven temperature mean that I will get the
optimum resolution of the heteroduplex and homoduplex species? 215
A3.3 I do not have the complete sequence of my fragment but I want
to scan for mutations. Is this still possible by DHPLC? 216
A3.4 Will DHPLC detect both heterozygous and homozygous
mutations? 217
A3.5 I have a sample population to scan for mutations and need
to be certain that I find all mutations present. What are the factors
affecting the accuracy of DHPLC and how should I approach the
problem? 217
A3.6 What are the minimum and maximum fragment sizes I can
analyze by DHPLC? 218
A3.7 I need to screen a large number of samples. What is the quickest
way to do this? 219
A3.8 I want to create a general SNP map but do not need to find
every mutation. What is the best strategy? 219
A3.9 What will happen if I have more than one mutation in a fragment?
Does each mutation or combination of mutations give a unique
chromatographic pattern? 220
A3.10 What about the converse? Does a particular chromatographic pattern
indicate a particular mutation? 220
A3.11 I have a biological system where it is likely that a mutation if
present can have a heterduplex concentration of less than 50 %.
What is my best approach to this problem? 221
A3.12 Is it possible to use DHPLC in a diagnostic setting? 222
References 223
Index 225
