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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

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

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 2530927 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

This book was carefully produced. Nevertheless, authors andpublisher 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 Acatalogue record for this publication is available from Die Deutsche Bibliothek. c WILEY-VCH Verlag GmbH, 69469 Weinheim (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 writtenpermission 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. Schaffer GmbH & Co. KG È ISBN 3-527-30244-1

CoverLascaux cover design by Atelier Prisma, Tatjana Treiber, 76344 Leopoldshafen, Germany

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 Introduction 1

1.3 1.4

General Background 1Short Historical Review of the Chromatography of Nucleic Acids 6 Terms and Definitions 8 Scope and Organization of This Book 10 References 11
Instrumentation and Operation 14

2 2.1 2.2 2.3 2.3.1 2.3.2 2.3.3 2.3.4 2.3.5 2.3.6 2.3.7 2.3.8 2.3.9 2.3.10 2.3.11 2.3.11.1 2.3.11.2 2.3.11.3 2.3.11.4

Introduction 14 General Description of the DNA Chromatograph 15 Detailed Description of the DNAChromatograph 16 The General Instrument and Materials 16 Dead Volume 17 Degassing the Eluent 18 Pumps 19 Gradient Formation 21 Pressure 23 Autosampler Injector 23 Separation Column 25 Column Protection 26 Column Oven 27 Detection 28 Selective vs. General Detection 28 Ultraviolet-Visible Detectors 29 Fluorescence Detector 31 Mass Spectrometry Detection 34

VI

Contents

2.3.12 2.3.12.1 2.3.12.22.3.12.3 2.3.13

Data Analysis 37 Size Analysis 38 Peak Shape or Pattern of Peaks 38 Quantification 39 Fragment Collection 40 References 41
Chromatographic Principles for DNA Separation 42

3 3.1 3.2 3.3 3.3.1 3.3.2 3.3.3 3.3.4 3.3.5 3.3.5.1 3.3.5.2 3.4 3.4.1 3.4.2 3.4.2.1 3.4.2.2 3.4.2.3 3.4.2.4 3.4.3 3.4.3.1 3.4.3.2 3.5 3.5.1 3.5.2 3.5.3 3.6 3.6.1 3.6.2 3.6.3

Introduction 42 Comparison of...