DNA Chromatography. Douglas T. Gjerde, Christopher P. Hanna, David Hornby
`Copyright c 2002 Wiley-VCH Verlag GmbH & Co. KGaA
`ISBNs: 3-527-30244-1 (Hardback); 3-527-60074-4 (Electronic)
`
`Douglas T. Gjerde
`Christopher P. Hanna
`David Hornby
`DNA Chromatography
`
`1
`
`MTX1027
`
`

`

`DNA Chromatography. Douglas T. Gjerde, Christopher P. Hanna, David Hornby
`Copyright c 2002 Wiley-VCH Verlag GmbH & Co. KGaA
`ISBNs: 3-527-30244-1 (Hardback); 3-527-60074-4 (Electronic)
`
`Douglas T. Gjerde, Christopher P. Hanna, David Hornby
`
`DNA Chromatography
`
`2
`
`

`

`DNA Chromatography. Douglas T. Gjerde, Christopher P. Hanna, David Hornby
`Copyright c 2002 Wiley-VCH Verlag GmbH & Co. KGaA
`ISBNs: 3-527-30244-1 (Hardback); 3-527-60074-4 (Electronic)
`
`Dr. Douglas T. Gjerde
`12295 Woodside Drive
`Saratoga, CA 95070
`USA
`Tel: 001-408 253 0927
`gjerde@earthlink.net
`
`Dr. Christopher P. Hanna
`c/o HTS Biosystems
`92 South Street
`Hopkinton, MA 01748
`USA
`Tel: 001-508 435 4700
`Channa8581@aol.com
`
`Dr. David Hornby
`Department of Molecular Biology
`University of Sheffield
`Western Bank
`Sheffield S10 2TN
`UK
`Tel: 0044-1 142 224 236
`Fax: 0044-1 142 762 687
`d.hornby@sheffield.ac.uk
`
`Cover
`Lascaux cover design by Atelier Prisma,
`Tatjana Treiber, 76344 Leopoldshafen,
`Germany
`
`This book was carefully produced.
`Nevertheless, authors and publisher do not
`warrant the information contained therein
`to be free of errors. Readers are advised to
`keep in mind that statements, data,
`illustrations, procedural details or other
`items may inadvertently be inaccurate.
`
`Library of Congress Card No.: applied for
`A catalogue record for this book is available
`from the British Library.
`
`Die Deutsche Bibliothek ±
`CIP Cataloguing-in-Publication-Data
`A catalogue record for this publication is
`available from Die Deutsche Bibliothek.
`
`c WILEY-VCH Verlag GmbH, 69469 Wein-
`heim (Federal Republic of Germany). 2002
`All rights reserved (including those of
`translation in other languages). No part of
`this book may be reproduced in any form ±
`by photoprinting, microfilm, or any other
`means ± nor transmitted or translated into
`machine language without written permis-
`sion from the publishers. Registered names,
`trademarks, etc. used in this book, even
`when not specifically marked as such, are
`not to be considered unprotected by law.
`
`Printed in the Federal Republic of Germany.
`
`Printed on acid-free paper.
`
`Typesetting Hagedorn Kommunikation,
`Viernheim
`Printing betz-druck gmbh, Darmstadt
`Bookbinding J. SchaÈffer GmbH & Co. KG
`
`ISBN 3-527-30244-1
`
`3
`
`

`

`DNA Chromatography. Douglas T. Gjerde, Christopher P. Hanna, David Hornby
`Copyright c 2002 Wiley-VCH Verlag GmbH & Co. KGaA
`ISBNs: 3-527-30244-1 (Hardback); 3-527-60074-4 (Electronic)
`
`V
`
`Contents
`
`Preface XI
`
`Acknowledgement XIII
`
`About the cover XV
`
`1
`1.1
`1.2
`
`1.3
`1.4
`
`Introduction 1
`General Background 1
`Short Historical Review of the Chromatography of
`Nucleic Acids
`6
`Terms and Definitions
`8
`Scope and Organization of This Book 10
`References
`11
`
`18
`
`Instrumentation and Operation 14
`2
`Introduction 14
`2.1
`General Description of the DNA Chromatograph 15
`2.2
`Detailed Description of the DNA Chromatograph 16
`2.3
`The General Instrument and Materials
`2.3.1
`16
`Dead Volume
`2.3.2
`17
`Degassing the Eluent
`2.3.3
`Pumps
`2.3.4
`19
`Gradient Formation 21
`2.3.5
`Pressure
`2.3.6
`23
`Autosampler Injector
`2.3.7
`23
`Separation Column 25
`2.3.8
`Column Protection 26
`2.3.9
`Column Oven 27
`2.3.10
`Detection 28
`2.3.11
`Selective vs. General Detection 28
`2.3.11.1
`2.3.11.2 Ultraviolet-Visible Detectors
`29
`Fluorescence Detector
`2.3.11.3
`31
`2.3.11.4 Mass Spectrometry Detection 34
`
`4
`
`

`

`VI Contents
`
`Data Analysis
`2.3.12
`37
`Size Analysis
`2.3.12.1
`38
`2.3.12.2 Peak Shape or Pattern of Peaks
`2.3.12.3 Quantification 39
`Fragment Collection 40
`2.3.13
`References
`41
`
`38
`
`50
`
`55
`
`57
`
`Chromatographic Principles for DNA Separation 42
`3
`Introduction 42
`3.1
`Comparison of Chromatography and Gel Electrophoresis 45
`3.2
`Basic Chromatographic Considerations
`3.3
`48
`Retention 49
`3.3.1
`Retention Factors
`3.3.2
`Peak Width 50
`3.3.3
`Plate Theory of Chromatography
`3.3.4
`51
`The Rate Theory of Chromatography
`3.3.5
`General Considerations
`3.3.5.1
`55
`Extra Column Effects
`3.3.5.2
`57
`Reverse Phase Column Packing Materials
`3.4
`Types of Materials
`3.4.1
`57
`Polymeric Resins
`3.4.2
`59
`Substrate and Crosslinking 59
`3.4.2.1
`Porous and Nonporous Resins
`3.4.2.2
`59
`3.4.2.3 Monolith Polymeric Columns
`62
`Functionalization of the Polymer
`3.4.2.4
`Silica-based Materials
`3.4.3
`64
`General Description 64
`3.4.3.1
`Functionalization 65
`3.4.3.2
`Reverse Phase Ion Pairing Chromatography
`3.5
`Principles
`3.5.1
`66
`Temperature Modes of DNA Chromatography
`3.5.2
`Effect of Metal Contamination 72
`3.5.3
`Ion Exchange Materials and Separation Mechanism 75
`3.6
`Polymer-based Anion Exchangers (Anex)
`3.6.1
`75
`Silica-based Anion Exchangers
`3.6.2
`77
`Basis for Separation 78
`3.6.3
`References
`79
`
`62
`
`66
`
`71
`
`4
`4.1
`4.2
`4.2.1
`4.2.2
`4.2.3
`4.2.4
`
`DHPLC 81
`Introduction 81
`Practice of the Technique
`83
`Melting Phenomena and Domains
`Temperature Prediction 85
`Primer Optimization and Clamping 88
`PCR Fidelity
`91
`
`83
`
`5
`
`

`

`Contents
`
`VII
`
`4.2.5
`4.2.5.1
`
`High-sensitivity DHPLC Determinations
`92
`Chromatographic Resolution between Heteroduplices
`and Homoduplices
`93
`4.2.5.2 Mass Sensitivity for the Resolved Heteroduplices
`PCR-induced Background 95
`4.2.5.3
`Example Application: Detection of Varying Levels of k-ras Alleles
`4.2.5.4
`Review of DHPLC Publications
`4.3
`98
`Conclusions
`4.4
`103
`References
`105
`
`93
`
`96
`
`5
`5.1
`5.2
`5.3
`5.4
`5.4.1
`5.4.2
`5.4.3
`
`6
`6.1
`6.2
`6.3
`6.4
`6.5
`6.5.1
`6.5.2
`6.6
`6.7
`
`7
`7.1
`7.2
`7.3
`7.4
`7.4.1
`7.4.2
`7.5
`7.6
`
`7.7
`7.7.1
`
`108
`
`108
`
`Size Based Separations
`Introduction 108
`Fundamental Developments
`Calibration 110
`Applications
`114
`Primer extension and near-size-based separations
`LOH and other size-based genotyping techniques
`Size Based Purification Procedures
`115
`References
`116
`
`114
`115
`
`118
`
`119
`
`Purification of Nucleic Acids
`Introduction 118
`System Dead Volume
`Cleaning 120
`Testing the Instrument Operation 122
`Calibration and Separation Conditions
`Internal and External Calibration 122
`Isocratic Elution 123
`Software Collection Methods
`Recovery of Material
`133
`References
`134
`
`122
`
`132
`
`135
`
`RNA Chromatography
`Introduction 135
`Biological Extraction of RNA 137
`Size Based RNA Separation 139
`Separation of Cellular RNA Species
`141
`Separation of Messenger RNA from Ribosomal RNA 141
`Analysis of Transfer RNA 143
`Chromatography and Analysis of Synthetic Oligoribonucleotides
`Application of RNA and DNA Chromatography in
`cDNA Library Synthesis
`149
`Analysis of Gene Expression by RNA and DNA Chromatography
`DNA Chromatography Analyses of RT-PCR and
`Competitive RT-PCR Products
`152
`
`145
`
`152
`
`6
`
`

`

`VIII Contents
`
`7.7.2
`7.7.3
`
`8
`8.1
`8.2
`8.3
`8.4
`8.4.1
`8.4.2
`8.4.3
`8.5
`
`8.6
`8.7
`
`Alternative Splicing 154
`Differential Messenger RNA Display via DNA Chromatography
`References
`158
`
`155
`
`160
`
`Special Techniques
`Introduction 160
`Analytical and Preparative Enzymatic Cleavage of DNA 161
`Analysis of DNA Methylation 164
`Nucleic Acid Enzymology
`171
`Telomerase Assays
`171
`Polynucleotide Kinase Assays
`173
`Uracil DNA Glycosylase Assays
`174
`Protein Nucleic Acid Interaction Mapping: ªFootprintingº
`Method Section 177
`Nucleic Acid Tagging 180
`DNA Chromatography with Intercalating Dyes
`References
`182
`
`175
`
`181
`
`9
`
`Looking Forward 184
`
`Appendix 1 Glossary of Terms
`
`187
`
`207
`
`Appendix 2 System Cleaning and Passivation Treatment
`Background Information 207
`A2.1
`Reagents
`A2.2
`211
`Preparation of the System 211
`A2.3
`Passivation of System 211
`A2.4
`Equilibration of System 212
`A2.5
`Passivation of Injection Port and Injection Needle
`A2.6
`References
`213
`
`212
`
`A3.3
`
`A3.4
`
`A3.5
`
`214
`Appendix 3 Frequently Asked DHPLC Questions
`What are the various methods for DHPLC temperature selection? 214
`A3.1
`Does having the optimum oven temperature mean that I will get the
`A3.2
`optimum resolution of the heteroduplex and homoduplex species? 215
`I do not have the complete sequence of my fragment but I want
`to scan for mutations. Is this still possible by DHPLC?
`216
`Will DHPLC detect both heterozygous and homozygous
`mutations? 217
`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
`What are the minimum and maximum fragment sizes I can
`analyze by DHPLC?
`218
`
`A3.6
`
`7
`
`

`

`Contents
`
`IX
`
`A3.7
`
`A3.8
`
`A3.9
`
`A3.10
`
`A3.11
`
`A3.12
`
`I need to screen a large number of samples. What is the quickest
`way to do this?
`219
`I want to create a general SNP map but do not need to find
`every mutation. What is the best strategy? 219
`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
`What about the converse? Does a particular chromatographic pattern
`indicate a particular mutation?
`220
`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
`Is it possible to use DHPLC in a diagnostic setting?
`References
`223
`
`222
`
`Index
`
`225
`
`8
`
`

`

`DNA Chromatography. Douglas T. Gjerde, Christopher P. Hanna, David Hornby
`Copyright c 2002 Wiley-VCH Verlag GmbH & Co. KGaA
`ISBNs: 3-527-30244-1 (Hardback); 3-527-60074-4 (Electronic)
`
`XI
`
`Preface
`
`The term DNA Chromatography is defined as a high performance, automatic
`separation and purification of DNA by high performance liquid chromatography
`(HPLC). Until recently, HPLC separation of DNA was too slow or DNA frag-
`ments were too poorly resolved to be of much use to the molecular biologist.
`Furthermore, most of the HPLC journal publications on DNA separation pub-
`lished by analytical chemists were written about technology that was not relevant
`to the needs of the researcher. For example, a typical publication might demon-
`strate the column and conditions to separate poly-T oligonucleotides up to 20
`nucleotides in length. Of course, demonstrating the separation of a mixture of
`poly-T oligonucleotide is of little interest
`to the molecular biologist because
`there is not much biological necessity to study this type of DNA. The analytical
`chemist simply did not understand the problems facing the molecular biologist.
`The analytical chemist knew that DNA separations were important, but did not
`understand how the molecular biologist needed to perform the separation or
`why and how the information would be used. This is not to disparage analytical
`chemists. It is only to say that there exists a chasm of understanding between
`analytical chemists and molecular biologists.
`This chasm must be crossed mainly because the needs of molecular biology
`science are changing rapidly. The tools that are needed to understand molecular
`biology are analytical tools. In order to understand and ultimately control the mo-
`lecular basis of life, analytical experiments must be designed and implemented,
`analytical tools must be used, and analytical information must be generated and
`studied. Because of its inherent analytical nature, DNA chromatography has the
`potential in becoming one of the leading analytical tools used by the molecular
`biologist.
`Who is to cross this chasm between analytical science and molecular biology?
`Some may say it must be the analytical chemist. They must explain the need for
`certain standards and methods for performing separations. But the onus cannot
`be completely on the chemist. The molecular biologist must recognize that there
`are new needs and standards that must be applied to their work. Thus, while
`the analytical chemist must teach their art to the molecular biologist by using
`terms that are clearly defined, the molecular biologist must teach the analytical
`chemist of their needs. Their needs are expressed in the objectives or goals of
`
`9
`
`

`

`XII
`
`Preface
`
`their research, how their experiments are designed and how the results might be
`used. It is the intent of the authors that by introducing DNA Chromatography, this
`book will provide some of the means to cross this chasm.
`
`Douglas T. Gjerde
`San Jose, California
`
`Chris Hanna
`Cambridge, Massachusetts
`
`David Hornby
`Sheffield, UK
`
`10
`
`

`

`DNA Chromatography. Douglas T. Gjerde, Christopher P. Hanna, David Hornby
`Copyright c 2002 Wiley-VCH Verlag GmbH & Co. KGaA
`ISBNs: 3-527-30244-1 (Hardback); 3-527-60074-4 (Electronic)
`
`XIII
`
`Acknowledgements
`
`The authors would like to acknowledge and thank many people who contributed
`or helped with the writing of this book. Leon Yengoyan of San Jose State
`University read major portions of this book and made many useful comments.
`The authors hope that they have used the comments and suggestions wisely.
`The authors are grateful to Joann Walters who made several helpful comments
`on Chapter 4 DHPLC and who also is the author of Appendix 3: Frequently
`Asked DHPLC Questions. The authors thank Maryam Matin and Mark Dickman
`who developed many of the RNA and DNA applications and helped assemble
`much of the information that is reported in this book. They are both extremely
`talented and the authors appreciate their dedication. The authors thank and
`acknowledge the contribution of Robert Haefele to Appendix 2, the section on
`system cleaning and passivation.
`Jeffrey Sklar of Harvard Medical School has provided many valuable insights re-
`garding DNA Chromatography technology as a whole, and all of the authors are
`grateful for his efforts through the years. The authors thank GuÈnther Bonn of
`Leopold-Franzens University Innsbruck for his support of the technology and for
`the many hours of discussions. Bert Volgelstein of Johns Hopkins Medical School
`provided the k-ras samples described in Chapter 4 and made very useful comments
`to the analyses that were performed.
`The authors are grateful to Arezou Azarani for the information provided on the
`denaturation of RNA and to John Brady and Bill Walker who were very helpful in
`providing many of the patent references and figures. Pam Smith and Tiffany
`Nguyen are thanked for their contributions to the organization of the literature
`and their help with the logistics of the project. The authors would like to thank
`their friends and colleagues for the many hours of enjoyable discussions.
`Finally, the authors would like to acknowledge and thank their families. They are
`the most important parts of our lives. Their support and encouragement did not go
`unnoticed and the authors express their love to them.
`
`11
`
`

`

`DNA Chromatography. Douglas T. Gjerde, Christopher P. Hanna, David Hornby
`Copyright c 2002 Wiley-VCH Verlag GmbH & Co. KGaA
`ISBNs: 3-527-30244-1 (Hardback); 3-527-60074-4 (Electronic)
`
`XV
`
`About the Cover
`
`The cave paintings found in France, Northern Spain and other places represent
`some of the earliest forms of enduring art produced by our species. They tell us
`about ourselves but they also prompt us to ask questions.
`Who were the people that made these paintings? At the dawn of civilization
`over 35,000 years ago it is possible Cro-Magnon and Neanderthal man lived at
`the same time? Did they interact to the extent that we are descendents of both
`species?
`Why are many of the animals depicted in the paintings now extinct? Is extinction
`something that man can avoid?
`One of the ways researchers are attempting to answer these questions is through
`DNA analysis. DNA is enduring. We are the same as the artists who painted these
`drawings. Our DNA will tell us our past, who we are and perhaps point the way to
`our future.
`
`12
`
`

`

`DNA Chromatography. Douglas T. Gjerde, Christopher P. Hanna, David Hornby
`Copyright c 2002 Wiley-VCH Verlag GmbH & Co. KGaA
`ISBNs: 3-527-30244-1 (Hardback); 3-527-60074-4 (Electronic)
`
`1
`
`1 I
`
`ntroduction
`
`1.1
`General Background
`
`The knowledge base of molecular biology has been built upon the use of analytical
`tools. Sequencing, allele specific amplification, quantitative PCR, ligase chain reac-
`tion, etc. are all analytical methods that, if used properly, allow molecular biologists
`to design experiments and interpret information and contribute to the base of
`knowledge. The success of any particular method depends on how well it can per-
`form its analytical function. How reproducible is the method? Does the method
`have interferences that could result in either false negative results or false positive
`results? Is the test robust? Will the analytical test function in the same manner
`every time it is used by an analyst or by several analysts?
`There has been rapid growth over the last ten years in the number of analytical
`tools available for the analysis of nucleic acids. The large majority of these tools are
`based or depend upon gel electrophoresis. More specifically, analytical methods
`have been developed around the assumption that gel electrophoresis is required
`for the development of the information being sought. Separation science is one
`of the mainstay tools used in chemistry and biology. In particular, technological ad-
`vances that have facilitated the analysis and preparation of pure biopolymers have
`been central to the development of modern molecular biology. Gel electrophoresis
`has allowed the separation and purification of a wide variety of nucleic acid mole-
`cules.
`Slab gel electrophoresis is the cornerstone tool of the molecular biologist. It has
`been in use for almost 60 years, and is on the lab bench of virtually all research-
`ers. The tool will separate DNA fragments and with visual scanning options, the
`researcher can determine the size and amount of DNA separated. High resolution
`purification of a particular band can be accomplished by cutting out with a scalpel
`or razor blade, then the particular band of interest is extracted from the gel
`matrix.
`As the speed of throughput in contemporary experimentation becomes ever
`more demanding, the need to automate has been acknowledged to be of prime im-
`portance. Due to many of the tedious aspects related to technique, there have been
`attempts to automate the slab gel electrophoresis process. There are very few op-
`
`13
`
`

`

`2
`
`1.1 General Background
`
`tions for simple sample introduction, separation, detection and automatic collec-
`tion of separated materials. Pre-cast gels with automated visual scanning detection
`devices have helped the technique.
`The introduction of capillary gel electrophoresis has been successful in circum-
`venting many of the drawbacks of slab gel electrophoresis. Materials separated by
`capillary gel electrophoresis are not easily purified and collected from the capil-
`lary instrument and therefore the instrument falls somewhat short as a suitable
`alternative for the molecular biologist.
`There will always be a strong need to analyze nucleic acids for purity and size
`and to prepare pure materials. In an effort to avoid the complex tasks of gel auto-
`mation, a variant of HPLC known as DNA Chromatography has been developed.
`HPLC is a highly automated technology. It employs automatic sample introduc-
`tion, separation, and detection and even automated collection of separated samples.
`Depending on the column and separation conditions, large quantities of nucleic
`acid fragments can be separated, purified and collected. The instrument is comput-
`er controlled.
`Simply stated, DNA Chromatography is the high performance separation of nu-
`cleic acids by high performance liquid chromatography (HPLC). High performance
`implies that modern standards of instrumentation are met, and separations of high
`purity are achieved. In the case of DNA, the HPLC instrumentation is computer
`controlled. The separation conditions are computed and implemented through
`input of the DNA sequence and the desired type of separation. DNA may be col-
`lected automatically for further downstream processing such as cloning, PCR, se-
`quencing, etc. The separations are performed usually in less than 10 minutes and,
`in many cases, single base pair resolution of the DNA is achieved.
`An example of an application of DNA Chromatography is illustrated in Figure 1.1.
`Competitive quantitative, reverse transcription PCR (Q-RT-PCR) is a sensitive
`method for measuring trace mRNA. A quantity of sample is mixed with a
`known amount of competitor to act as an internal standard. The competitor has
`almost an identical sequence to the unknown except for an (approximately 20
`base) insertion somewhere in the fragment. The two species are reverse tran-
`scribed to cDNA and the sample is amplified by PCR (at equal efficiency for sam-
`ple mRNA and competitor). The concentration of the sample mRNA is calculated
`using the concentration of the competitor standard and the fragment measure-
`ments. Previously, the measurement of the fragments had been done by gel elec-
`trophoresis analysis. Peter Doris and his coworkers worked out the method to per-
`form the analysis by DNA Chromatography [1, 2]. They found that this allowed a
`much more accurate measurement than previous methods mainly because the het-
`eroduplex complexes of the sample and competitor could now be reliably resolved.
`Gel methods usually don't have the resolution to separate the heteroduplex species
`from the sample and competitor fragments leading to errors in the calculation of
`the mRNA concentration. The DNA Chromatography method can be applied to
`high-throughput research and clinical facilities, especially where high sensitivity
`(for low expressed forms of mRNA) and high accuracy of mRNA measurements
`are needed. (Details of the method are described in Chapter 7).
`
`14
`
`

`

`1 Introduction
`
`3
`
`Figure 1.1. Separation of Q-RT-PCR target
`204 bp sample fragment, 228 bp competitor
`DNA fragment, and heteroduplex on a
`DNASepr column and WAVEr System: Eluent
`
`A: 0.1 M triethylammonium acetate, pH 7.0
`(TEAA); B: 0.1 M TEAA, 25 % acetonitrile (from
`Ref. [2] with permission).
`
`There have been many different approaches published on the analysis and pu-
`rification of nucleic acids by chromatography. A short historical review is described
`in the next section. The fundamental technology leading to modern DNA Chroma-
`tography was first described by GuÈnther Bonn, Christian Huber and Peter Oefner
`in 1993 [35]. They showed rapid, high resolution separations of both double-
`stranded and single-stranded DNA. The separations were performed usually in
`less than 10 minutes and, in many cases, single base pair resolution was achieved.
`This form of HPLC analysis is largely (though not entirely) based upon the unique
`separation properties of a non-porous polystyrenedivinylbenzene polymer bead
`that has been functionalized with C-18 alkyl groups. An alkylammonium salt is
`added to the eluent and forms neutral ion pairs when a DNA sample is introduced
`into the HPLC instrument. A gradient of acetonitrile solvent separates the DNA
`fragment with the smaller fragments coming off the column first and then larger
`fragments eluting off the column and traveling through the detector. Figure 1.2
`shows a separation of double-stranded DNA that can be achieved.
`The separation occurs in such a manner that classic gel-based separation are
`mimicked. Bonn, Huber and Oefner showed that the DNA separations were per-
`formed according to the size of the fragment just are they are in gel electropho-
`resis. For double-stranded DNA, the sequence does not contribute to the retention
`of the fragment. Figure 1.3 taken from their work demonstrates this with a plot of
`retention time vs. fragment size for a number of different fragments. Various plas-
`
`15
`
`

`

`4
`
`1.1 General Background
`
`Figure 1.2. High performance DNA Chroma-
`tography separation of double-stranded DNA
`using a DNASepr column 50 x 4.6 mm. Sample
`was a mixture of BR322 Hae III restriction di-
`gest and _174 Hinc II restriction digest. Eluent
`
`and Gradient: A: 0.1 M TEAA; B: 0.1 M TEAA,
`25 % acetonitrile, 35 to 45 % B in 2 min., 45 %
`to 57 % in 10 min., 57 % to 61 % in 4 min., 1.0
`mL/min flow rate, UV detection at 254 nm
`(from Ref. [4] with permission).
`
`Figure 1.3. The retention times of various
`plasmid digest fragments are plotted according
`to size. The fragments have different se-
`quences, but are separated according to their
`size using a DNASepr column 50 x 4.6 mm.
`
`Eluent and Gradient: A: 0.1 M TEAA; B: 0.1 M
`TEAA, 25 % acetonitrile, 0 % to 100 % B in
`30 min., 1.0 mL/min flow rate, UV detection at
`254 nm (from Ref. [41] with permission).
`
`16
`
`

`

`1 Introduction
`
`5
`
`mids were digested with a number of different enzymes so that a mixture of DNA
`fragments were generated. Since the sequence of the plasmids are known, the ef-
`fect of DNA sequence can be shown. In the plot, all of the retention times of the
`various fragments fall on a line drawn through the data. This shows that the reten-
`tion of DNA is dependent on fragment size, and is independent of fragment se-
`quence.
`One major difference between chromatography and electrophoresis is the use of
`gradient elution in chromatography. The content of the fluid (eluent) that is
`pumped through the column can be changed in real time as the separation is pro-
`ceeding. This is called a gradient process whereby the ªstrengthº or ªeluting
`powerº of the eluent is increased as the separation is developed. Small fragments
`are eluted first and then the organic solvent is increased and larger and larger frag-
`ments are eluted. The time required for the entire separation depends on the
`ªeluting programº that is employed. A rapid increase in the stronger eluent will
`elute the fragments faster. But if a rapid program is used, a separation with
`lower resolution of peaks could result. Conversely, a slow gradient process will pro-
`duce the highest resolving conditions and greatest separation of peaks. However,
`this carries the penalty of a longer separation time. If the DNA Chromatographic
`system is well designed and a high efficiency column is used, then rapid gradients
`will produce suitable and useful separations.
`Conversely, electrophoresis must rely on the conditions selected for the entire
`separation. The resolving conditions are not changed once the run is initiated;
`thus, whatever conditions were first selected must be used for the entire process.
`The ability to separate with a fluid gradient gives DNA Chromatography a tremen-
`dous advantage over gel electrophoresis.
`Another advantage of DNA Chromatography is the ability to detect the DNA
`fragment without fluorescence detection. Tags need not be used and the fragments
`may be detected directly at the sub nanogram level by UV automatic detection. Of
`course fluorescence detection could also be used provided fluorescence tags are
`added to the DNA. Use of this detection method decreases the amount of DNA
`that can be detected by a factor of 10 to 100 (or even 1000 in some reported cases).
`The use of column temperature to control separations in DNA chromatography
`was described in 1996 through the insights of Oefner and Underhill [69]. They
`demonstrated that DNA Chromatography possessed unique properties enabling
`the separation of DNA based on its relative degree of helicity. Heteroduplex
`DNA has a lower melting point than homoduplex DNA. The retention of single-
`stranded DNA is lower on the column than double-stranded DNA, thus heterodu-
`plex DNA melts more easily, consequently it comes off or elutes from the column
`earlier in the separation. This technique is called denaturing HPLC (DHPLC). In
`short, they developed a method of DNA chromatography that is analogous to dena-
`turing gradient gel electrophoresis (DGGE).
`Gel electrophoresis is a powerful tool. But, depending on the problem, DNA
`Chromatography has many advantages. The development of high resolution chro-
`matographic methods to replace many of the tasks now performed by gel electro-
`phoresis almost seems inevitable.
`
`17
`
`

`

`6
`
`1.2 Short Historical Review of the Chromatography of Nucleic Acids
`
`1.2
`Short Historical Review of the Chromatography of Nucleic Acids
`
`Much of the early liquid chromatography work published on the separation and
`purification of nucleic acids may seem crude by comparison to modern capabil-
`ities. But it would be a mistake to completely discount this early work. Many of
`the research reports describe sample preparation schemes, experimental design,
`separation schemes, and down stream processing of materials that are still impor-
`tant. Certainly, the advanced technologies in use today are built upon the careful
`work that preceded these advances. Still other early work is remarkable in its fore-
`sight and quality. The short review in this section is not meant to be complete and
`only some of the important references are cited. The section is intended to give a
`brief overview of the types of work that have been performed.
`The development of the high performance separation of nucleic acids has had a
`long history beginning with the early days of liquid chromatography. The progress
`has proceeded at a slow pace even as high quality separations of other biomolecules
`such as proteins were being developed. DNA Chromatography has unique require-
`ments for clean instrumentation and well designed columns and cleaning methods
`as described in this book. But single-stranded DNA has less stringent requirements
`than double-stranded DNA, and the best publications were based on work per-
`formed with single-stranded DNA.
`J. Thompson and coworkers published many excellent papers both in the form of
`review articles [1015] and research papers [1618]. The review articles published
`in 1986 and 1987 are a series of 6 publications, each dealing with some aspect of
`nucleic acid separation. In the first review (I) paper, Thompson presented a brief
`overview of the development of chromatographic stationary phases for liquid chro-
`matography. He stated that parallel improvements in both silica and polymer-based
`resins were helping to change liquid chromatography nucleic acid separations
`from classical techniques to more advanced HPLC techniques. He discussed
`many different types of chromatographic separations, (some of which will be dis-
`cussed in this section), ion exchange, and ion-pairing, reverse phase separations
`(the basis for much of this book). Reviews II, III, and IV discussed the isolation,
`purification, and analysis of single-stranded DNA, plasmids and double-stranded
`DNA. The separations are quite good, but still mostly slower, and lower resolution
`than what can be achieved today.
`Review V describes the use of affinity chromatography. Double-stranded nucleic
`acid is held together by hydrogen bonding of the two pyrimidine bases; thymine
`(T), and cytosine (C) and the two purine bases; adenine (A) and guanine (G).
`The basis for forming the double-stranded DNA is that sequence A will always hy-
`drogen bond with T, and G will always hydrogen bond with C. In affinity chroma-
`tography, a short single-stranded of DNA is attached to a solid support (perhaps 30
`bases). The sample containing the target nucleic acid fragment is passed through a
`column containing the support. Only the sample nucleic acid that finds an exact
`match with the complementary fragment will become attached to the solid support
`through hydrogen bonding. Other sample matrix nucleic acids and sample matrix
`
`18
`
`

`

`1 Introduction
`
`7
`
`materials are washed through the column. And then using solvent or heat, the tar-
`get nucleic acid fragment is denatured from the support and collected. An example
`of this technology is a support containing a bound oligo (dT) polymer used to pu-
`rify mRNA through its poly A tail. Much of the review discusses the various sup-
`ports to which the binder nucleic acid is attached including cellulose, nitrocellu-
`lose, methylated cellulose, cellulose acetate, and of course silica and polymers.
`The review also discussed the conditions for hybridization and denaturation of
`the target material.
`There are many proteins that are attracted to specific DNA sequences. In Review
`VI, Thompson et al discussed using the same sort of substrates to study DNA-bind-
`ing proteins. Many proteins that function in association with cellular nucleic acid
`recognize these polymer as a substrate and bind tightly under various conditions.
`Binding under different variables such as different ligand recognition sequences,
`ionic strength, pH, divalent cations and non-specific competitior DNA can be
`studied to determine the strength of the binding.
`There have been many studies of different ways to separate DNA and RNA. A
`few of these studies are listed here. Size exclusion of double-stranded DNA restric-
`tion fragments was shown to be an accurate method for size determination of un-
`known DNA. The separation is based on a hydrated gel packing particles. Smaller
`fragments can penetrate the column packing and travel through the column more
`slowly. Larger fragments are excluded from the packing and travel through the col-
`umn faster. The separation is the inverse to gel electrophoresis where the basis for
`sepa