Molecular Diagnostics, Notas de estudo de Biomedicina

Baixe Molecular Diagnostics e outras Notas de estudo em PDF para Biomedicina, somente na Docsity! George P. Patrinos Wilhelm Ansorge Rose Diagnostics E) |  Molecular Diagnostics Academic Press is an imprint of Elsevier 32 Jamestown Road, London NW1 7BY, UK 30 Corporate Drive, Suite 400, Burlington, MA 01803, USA 525 B Street, Suite 1900, San Diego, CA 92101-4495, USA First edition 2005 Second edition 2010 Copyright © 2010 Elsevier Ltd. All rights reserved No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the publisher. Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone (  44) (0) 1865 843830, fax (  44) (0) 1865 853333; email: permissions@elsevier.com . 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Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress ISBN : 978-0-12-374537-8 For information on all Academic Press publications visit our website at www.elsevierdirect.com Typeset by Macmillan Publishing Solutions ( www.macmillansolutions.com ) Printed and bound in the United States of America 10 11 12 13 14 15 10 9 8 7 6 5 4 3 2 1 v Contents List of Contributors ix Preface – First Edition xiii Preface – Second Edition xv Foreword – First Edition xvii 1. Molecular Diagnostics: Past, Present, and Future 1 George P. Patrinos and Wilhelm J. Ansorge Section I Molecular Diagnostic Technology 13 2. Allele-Specifi c Mutation Detection 15 Aglaia Athanassiadou, Eleana F. Stavrou, Adamandia Papachatzopoulou and George P. Patrinos 3. Enzymatic and Chemical Cleavage Methods to Identify Genetic Variation 29 Chinh T. Bui, Emmanuelle Nicolas, Georgina Sallmann, Maria Chiotis, Andreana Lambrinakos, Kylee Rees, Ian Trounce, Richard G.H. Cotton, Lauryn Blakesley, Andrew K. Godwin and Anthony T. Yeung 4. Mutation Detection by Single Strand Conformation Polymorphism and Heteroduplex Analysis 45 Panayiotis G. Menounos and George P. Patrinos 5. Capillary Electrophoresis 59 Anja Bosserhoff and Claus Hellerbrand 6. Temperature and Denaturing Gradient Gel Electrophoresis 75 Hartmut Peters and Peter N. Robinson 7. Real-Time Polymerase Chain Reaction 87 Lut Overbergh, Anna-Paula Giulietti, Dirk Valckx and Chantal Mathieu 8. Pyrosequencing 107 Sharon Marsh 9. Application of Padlock and Selector Probes in Molecular Medicine 117 Mats Nilsson, Chatarina Larsson, Johan Stenberg, Jenny G ö ransson, Ida Grundberg, Magnus Isaksson, Tim Conze and Sara Henriksson 10. Molecular Cytogenetics in Molecular Diagnostics 133 Holger T ö nnies 11. Analysis of Human Splicing Defects Using Hybrid Minigenes 155 Franco Pagani and Francisco E. Baralle 12. Detection of Genomic Duplications and Deletions 171 Graham R. Taylor and Carol A. Delaney 13. Multiplex Ligation-Dependent Probe Amplifi cation (MLPA) and Methylation- Specifi c (MS)-MLPA: Multiplex Detection of DNA/mRNA Copy Number and Methylation Changes 183 Eline M. Sepers and Jan P. Schouten 14. Molecular Techniques for DNA Methylation Studies 199 J ö rg Tost and Ivo G. Gut 15. High-Resolution Melting Curve Analysis for Molecular Diagnostics 229 Jared S. Farrar, Gudrun H. Reed and Carl T. Wittwer 16. DNA Microarrays and Genetic Testing 247 Lars Dyrskj ø t, Karina Dalsgaard-S ø rensen, Marie Stampe-Ostenfeld, Karin Birkenkamp- Demtroder, Kasper Thorsen, Claus L. Andersen, Mogens Kruh ø ffer, Jens L. Jensen and Torben F. Ø rntoft Contentsvi 17. Arrayed Primer Extension Microarrays for Molecular Diagnostics 267 Neeme Tonisson, Eneli Oitmaa, Kaarel Krjutskov, Janne Pullat, Ilona Lind, Merike Leego, Ants Kurg and Andres Metspalu 18. Application of Proteomics to Disease Diagnostics 285 Samir Hanash, Ji Qiu and Vitor Faca 19. RNA-Based Variant Detection: The Protein Truncation Test 293 Johan T. den Dunnen 20. Protein Diagnostics by Proximity Ligation: Combining Multiple Recognition and DNA Amplifi cation for Improved Protein Analyses 299 Karl -Johan Leuchowius, Ola S ö derberg, Masood Kamali-Moghaddam, Malin Jarvius, Irene Weibrecht, Katerina Pardali and Ulf Landegren 21. Mass Spectrometry and its Applications to Functional Proteomics 307 Thomas K ö cher and Giulio Superti-Furga Section II Applications of Molecular Diagnostics and Related Issues 325 22. Pharmacogenetics and Pharmacogenomics: Impact on Drug Discovery and Clinical Care 327 Minoli Perera and Federico Innocenti 23. Nutrigenomics: Integrating Genomic Approaches into Nutrition Research 347 Lynette R. Ferguson, Martin Philpott and Matthew P.G. Barnett 24. Novel Next-Generation DNA Sequencing Techniques for Ultra High-Throughput Applications in Bio-Medicine 365 Wilhelm J. Ansorge 25. Locus-Specifi c and National/Ethnic Mutation Databases: Emerging Tools for Molecular Diagnostics 379 George P. Patrinos 26. Molecular Diagnostic Applications in Forensic Science 393 Bruce Budowle, John V. Planz, Rowan Campbell and Arthur J. Eisenberg 27. Mass Disaster Victim Identifi cation Assisted by DNA Typing 407 Daniel Corach 28. Detection of Highly Pathogenic Viral Agents: Implications for Therapeutics, Vaccines and Biodefense 417 Kylene Kehn-Hall and Sina Bavari 29. Identifi cation of Genetically Modifi ed Organisms 431 Farid E. Ahmed 30. Molecular Diagnostics and Comparative Genomics in Clinical Microbiology 445 Alex van Belkum 31. Genetic Monitoring of Laboratory Rodents 461 Jean -Louis Gu é net and Fernando J. Benavides 32. Safety Analysis in Retroviral Gene Therapy: Identifying Virus Integration Sites in Gene-Modifi ed Cells 471 Stephanie Laufs, Luisa Schubert, Patrick Maier, W. Jens Zeller and Stefan Fruehauf 33. Preimplantation Genetic Diagnosis 485 Martine de Rycke and Karen Sermon 34. Automated DNA Hybridization and Detection 501 Bert Gold 35. The Use of Microelectronic-Based Techniques in Molecular Diagnostic Assays 513 Piotr Grodzinski, Michael Ward, Robin Liu, Kathryn Scott, Saul Surrey and Paolo Fortina 36. Human Gene Patents and Genetic Testing 527 Timothy Caulfi eld and Yann Joly ix Contributors Prof . Dr. Farid E. Ahmed Professor , Department of Radiation Oncology, Leo W. Jenkins Cancer Center, East Carolina University, The Brody School of Medicine, 600 Moye Boulevard, LSB 014, Greenville, NC 27858, USA. Dr . Claus L. Andersen Associate Professor, Molecular Diagnostic Laboratory, Department of Clinical Biochemistry, Aarhus University Hospital, Skejby, 8200-Aarhus N, Denmark. Prof . Dr. Wilhelm J. Ansorge Visiting Professor, Ecole Polytechnique Federal Lausanne, PH D2 445 (B â timent PH) Station 3, Lausanne, CH-1015, Switzerland . Prof . Dr. Aglaia Athanassiadou Professor of General Biology, University of Patras, Faculty of Medicine, School of Health Sciences, Department of General Biology, Asclepiou Street, Panepistimioupolis, GR- 26110, Patras, Greece. Prof . Dr. Francisco E. Baralle Head , Molecular Pathology Laboratory; Director, International Centre for Genetic Engineering and Biotechnology (ICGEB), Padriciano, 99, 34012 Trieste, Italy. Dr . Matthew P. G. Barnett Senior Research Scientist, Food, Metabolism and Microbiology, Food and Textiles Group, AgResearch Limited, Palmerston North, New Zealand. Dr . Sina Bavari Chief of Immunology, Target Identification, and Translational Research, United States Army Medical Research Institute of Infectious Diseases, Bacteriology Division, Ft. Detrick, MD, 21702, USA. Prof . Dr. Alex van Belkum Professor of Molecular Microbiology, Erasmus MC, Department of Medical Microbiology and Infectious Diseases, Unit Research and Development, Dr. Molewaterplein 40, 3015 GD Rotterdam, The Netherlands. Dr . Walter Bell Associate Professor of Pathology, Division of Anatomic Pathology, University of Alabama at Birmingham, North Pavilion – Room 3540, University Station, Birmingham, AL 35294-7331, USA. Dr . Fernando Benavides Associate Professor and Director, Genetic Services, Department of Carcinogenesis, The University of Texas MD Anderson Cancer Center, 1808 Park Road 1C – PO Box 389, Smithville, TX 78957, USA. Dr . Karin Birkenkamp-Demtroder Associate Professor, Molecular Diagnostic Laboratory, Department of Clinical Biochemistry, Aarhus University Hospital, Skejby, 8200-Aarhus N, Denmark. Lauryn Blakesley Research Assistant, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111- 2497, USA. Prof . Dr. Anja Katrin Bosserhoff Professor of Molecular Pathology, University of Regensburg, Institute of Pathology, Franz-Josef-Strau ß -Allee 11, D-93042 Regensburg, Germany. Dr . Bruce Budowle Senior Scientist, Federal Bureau of Investigation, Laboratory Division, 2501 Investigation Parkway, Quantico, Virginia, 22135, USA. Dr . Chinh Thien Bui Senior Research Officer, Walter and Eliza Hall Research Institute, 4 Research Ave, Latrobe Research and Development Park, Bundoora, Victoria 3086, Australia. Dr . David Burnett Consultant in Quality and Accreditation Systems, Lindens Lodge, Bradford Place, Penarth, CF64 1LA, United Kingdom. Dr . Rowan S. Campbell Research Associate, University of North Texas, School of Medicine, PO Box 311277, Denton, Texas 76203, USA. Prof . Dr. Timothy Caulfield Canada Research Chair in Health Law and Policy, Research Director, Health Law Institute; Professor, Faculty of Law, Faculty of Medicine and Dentistry, 461 Law Centre, University of Alberta, Edmonton, Alberta, T6G 2H5, Canada. Maria Chiotis Research Assistant, Genomic Disorders Research Centre, 7th Floor, Daly Wing, St. Vincent’s Hospital, 41 Victoria Parade, Fitzroy 3065, Melbourne, Victoria, Australia. Tim Conze Graduate Student, Department of Genetics and Pathology, Uppsala University, Rudbeck Laboratory, SE-751 85, Uppsala, Sweden. Prof . Dr. Daniel Corach Professor , Department of Genetics and Molecular Biology, University of Buenos Aires, School of Pharmacy and Biochemistry; Director, DNA Fingerpring Service (Servicio de Hiellas Digitales Gen é ticas-SHDG); Principal Research Fellow, National Research Council (CONICET), Junin 956, 1113- Buenos Aires, Argentina. Prof . Dr. Richard G.H. Cotton Director , Genomic Disorders Research Centre, 7th Floor, Daly Wing, St. Vincent’s Hospital, 41 Victoria Parade, Fitzroy 3065, Melbourne, Victoria, Australia. Dr . Karina Dalsgaard-S ø rensen Associate Professor, Molecular Diagnostic Laboratory, Department of Clinical Biochemistry, Aarhus University Hospital, Skejby, 8200-Aarhus N, Denmark. Dr . Carol A. Delaney Clinical Scientist in Molecular Cytogenetics, SE Scotland Cytogenetics Laboratory, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, United Kingdom. Contributorsx Dr . Martine de Rycke Senior Scientist, Centre for Medical Genetics, UniversitairZiekenhuis Brussel, Department of Embryology and Genetics, Vrije Universiteit Brussel, Laarbeeklaan 101, B-1090 Brussels, Belgium. Johan T. den Dunnen Leiden Genome Technology Center, Human and Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands. Dr . Lars Dyrskj ø t Associate Professor, Molecular Diagnostic Laboratory, Department of Clinical Biochemistry, Aarhus University Hospital, Skejby, 8200-Aarhus N, Denmark. Dr . Arthur J. Eisenberg Associate Professor and Director, DNA Identity Laboratory, Department of Pathology and Anatomy, University of North Texas Health Science Center, 3500 Camp Bowie Boulevard, Ft. Worth, Texas 76107, USA. Dr . Vitor Faca Post -Doctoral Fellow, Department of Molecular Diagnostics, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue N, M5-C800, Seattle, WA 98109, USA. Jared S. Farrar Laboratory Assistant, University of Utah School of Medicine, Department of Pathology, 50 N Medical Drive, Salt Lake City, UT 84132, USA. Prof . Dr. Lynnette R. Ferguson Programme Leader, Nutrigenomics New Zealand; Professor and Head, Discipline of Nutrition, Faculty of Medical and Health Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand. Prof . Dr. Paolo Fortina Professor of Medicine, Thomas Jefferson University; Director of Genomics and Diagnostics Center for Translational Medicine, Department of Medicine, 408 College Building, 1025 Walnut Street, Philadelphia, PA 19107, USA. Dr . Jerry Fredenburgh Chief Executive Officer, Fredenburgh Consultants, 3403 Cypress Drive, Spring Grove, IL 60081-8617, USA. Prof . Dr. Stefan Fruehauf Professor , Center for Tumor Diagnostics and Therapy, Paracelsus-Klinik, Am Natruper Holz 69, D-49090 Osnabr ü ck, Germany. Dr . Anna-Paula Giulietti Scientific Researcher, Laboratory for Experimental Medicine and Endocrinology (LEGENDO), Catholic University of Leuven, UZ Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium. Dr . Andrew K. Godwin Member , Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111- 2497, USA. Dr . Bert Gold Staff Scientist, Human Genetics Section, Laboratory of Genomic Diversity, National Cancer Institute at Frederick, Building 560, Room 21-21, Frederick, MD, 21702, USA. Jenny G ö ransson Graduate Student, Department of Genetics and Pathology, Uppsala University, Rudbeck Laboratory, SE-751 85, Uppsala, Sweden. Prof . Dr. William E. Grizzle Professor of Pathology, Department of Pathology, University of Alabama at Birmingham, Zeigler Research Building – Room 408, 703 South 19th Street Birmingham, AL 35294-0007, USA. Dr . Piotr Grodzinski Center for Strategic and Scientific Initiative, National Cancer Institute, Bethesda, MD, USA. Ida Grundberg Graduate Student, Department of Genetics and Pathology, Uppsala University, Rudbeck Laboratory, SE-751 85, Uppsala, Sweden. Dr . Jean-Louis Gu é net Emeritus Scientist, Institut Pasteur, France. Dr . Ivo G. Gut Assistant Director and Department Head, Department for Translational Research, Centre National de G é notypage, CEA – Institut de G é nomique, 2 rue Gaston Cr é mieux, F-91000 Evry Cedex, France. Prof . Dr. Samir Hanash Professor and Head, Department of Molecular Diagnostics, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue N, M5- C800, Seattle, WA 98109, USA. Dr . Claus Hellerbrand Assistant Professor, University Hospital Regensburg, Department of Internal Medicine I, Franz-Josef-Strau ß -Allee 11, D-93042 Regensburg, Germany. Sara Henriksson Graduate Student, Department of Genetics and Pathology, Uppsala University, Rudbeck Laboratory, SE-751 85, Uppsala, Sweden. Dr . Federico Innocenti Assistant Professor, University of Chicago, Department of Medicine, Section of Hematology/Oncology, 5841 South Maryland Avenue, MC 2115, Chicago, IL 60637, USA. Magnus Isaksson Graduate Student, Department of Genetics and Pathology, Uppsala University, Rudbeck Laboratory, SE-751 85, Uppsala, Sweden. Malin Jarvius Graduate Student, Department of Genetics and Pathology, Uppsala University, Rudbeck Laboratory, SE-751 85, Uppsala, Sweden. Prof . Dr. Jens L. Jensen Professor , Molecular Diagnostic Laboratory, Department of Clinical Biochemistry, Aarhus University Hospital, Skejby, 8200-Aarhus N, Denmark Yann Joly Research Scientist, University of Montreal, Public Law Research Center, Montr é al, Qu é bec, Canada. Dr . Masood Kamali-Moghaddam Associate Professor, Department of Genetics and Pathology, Uppsala University, Rudbeck Laboratory, SE-751 85, Uppsala, Sweden. Dr . Kylene Kehn-Hall Assistant Research Professor, Department of Microbiology, Immunology, and Tropical Medicine, The George Washington University, 2300 I Street, NW Ross Hall, Room 551, Washington, DC 20037, USA. Thomas K ö cher IMP – Research Institute of Molecular Pathology, Vienna, Austria. Kaarel Krjutskov Institute of Molecular and Cell Biology, University of Tartu/Estonian Biocentre, Tartu, Estonia and Asper Biotech, Tartu, Estonia. Dr . Mogens Kruh ø ffer Associate Professor, Molecular Diagnostic Laboratory, Department of Clinical Contributors xi Biochemistry, Aarhus University Hospital, Skejby, 8200-Aarhus N, Denmark. Ants Kurg Institute of Molecular and Cell Biology, University of Tartu/Estonian Biocentre, Tartu, Estonia and Asper Biotech, Tartu, Estonia. Andreana Lambrinakos Ph .D. Scholar, Genomic Disorders Research Centre, 7th Floor, Daly Wing, St. Vincent’s Hospital, 41 Victoria Parade, Fitzroy 3065, Melbourne, Victoria, Australia. Prof . Dr. Ulf Landegren Professor , Department of Genetics and Pathology, Uppsala University, Rudbeck Laboratory, SE-751 85, Uppsala, Sweden. Chatarina Larsson Graduate Student, Department of Genetics and Pathology, Uppsala University, Rudbeck Laboratory, SE-751 85, Uppsala, Sweden. Dr . Stephanie Laufs Research Scientist, Department of Experimental Surgery, Medical Faculty Mannheim, University of Heidelberg, and Department of Molecular Oncology of Solid Tumors Unit, German Cancer Research Center (DKFZ), Heidelberg, Germany; Deutsches Krebsforschungszentrum (DKFZ), G360 Klinische Kooperationseinheit Molekulare Onkologie Solider Tumoren, Im Neuenheimer Feld 580, 69120 Heidelberg, Germany. Merike Leego Institute of Molecular and Cell Biology, University of Tartu/Estonian Biocentre, Tartu, Estonia and Asper Biotech, Tartu, Estonia. Karl -Johan Leuchowius Graduate Student, Department of Genetics and Pathology, Uppsala University, Rudbeck Laboratory, SE-751 85, Uppsala, Sweden. Ilona Lind Asper Biotech, Tartu, Estonia and Tartu University Hospital, Estonia. Dr . Robin Liu Molecular Diagnostics, Osmetech Molecular Diagnostics, Pasadena, CA, USA. Dr . Patrick W. Maier Research Scientist, Department of Radiation Oncology, Mannheim Medical Centre, University of Heidelberg; Universit ä tsklinik f ü r Strahlentherapie und Radioonkologie, Univer- sit ä tsklinikum Mannheim, Universit ä t Heidelberg, Theodor-Kutzer-Ufer 1-3, D-68167 Mannheim, Germany. Sharon Marsh G é nome Qu é bec and Montreal Heart Institute Pharmacogenomics Centre, Montreal, Quebec, Canada. Dr . Chantal Mathieu Associate Professor of Medicine, Laboratory for Experimental Medicine and Endocrinology (LEGENDO), Catholic University of Leuven, UZ Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium. Dr . Panayiotis G. Menounos Director , Laboratory of Research, Nursing Military Academy, SAKETA ‘ ‘ A ’ ’ barrack, Vironas GR-16201, Athens, Greece . Andres Metspalu Institute of Molecular and Cell Biology, University of Tartu/Estonian Biocentre, Tartu, Estonia, Asper Biotech, Tartu, Estonia and Estonian Genome Project, University of Tartu. Dr . Emmanuelle Nicolas Research Associate, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111-2497, USA. Dr . Mats Nilsson Associate Professor, Uppsala University, Department of Genetics and Pathology, Rudbeck Laboratory, SE-751 85, Uppsala, Sweden. Eneli Oitmaa Institute of Molecular and Cell Biology, University of Tartu/Estonian Biocentre, Tartu, Estonia and Asper Biotech, Tartu, Estonia. Prof . Dr. Torben F. Ø rntoft Professor , Molecular Diagnostic Laboratory, Department of Clinical Biochemistry, Aarhus University Hospital, Skejby, 8200-Aarhus N, Denmark. Dr . Lut Overbergh Scientific Researcher, Laboratory for Experimental Medicine and Endocrinology (LEGENDO), Catholic University of Leuven, UZ Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium. Dr . Franco Pagani Staff Scientist, International Centre for Genetic Engineering and Biotechnology (ICGEB), Padriciano, 99, 34012 Trieste, Italy. Dr . Adamandia Papachatzopoulou Assistant Professor, University of Patras, Faculty of Medicine, School of Health Sciences, Department of General Biology, Asclepiou Street, Panepistimioupolis, GR-26110, Patras, Greece. Dr . Katerina Pardali Post -Doctoral Fellow, Department of Genetics and Pathology, Uppsala University, Rudbeck Laboratory, SE-751 85, Uppsala, Sweden. Dr . Andrea Farkas Patenaude Director of Psycho- Oncology Research and Assistant Professor of Psychology, Division of Pediatric Oncology, Dana-Farber Cancer Institute; Department of Psychiatry, Harvard Medical School, 44 Binney Street, Boston MA 02445, USA. Dr . George P. Patrinos Assistant Professor and Head, Pharmacogenomics Group, University of Patras, School of Health Sciences, Department of Pharmacy University Campus, GR-26504, Rion, Patras, Greece; Adjunct Assistant Professor, Erasmus University Medical Cen- ter, Faculty of Medicine and Health Sciences, Department of Bioinformatics, Room Ee1579, PO Box 2040, 3000 CA, Rotterdam, the Netherlands. Dr . Minoli Perera Instructor , University of Chicago, Department of Medicine, Section of Genetic Medicine, 5841 South Maryland Ave, Room TS 651, MC 6091, Chicago IL, 60637, USA . Dr . Hartmut Peters Research Scientist, Institute of Medical Genetics, Charit é -Universit ä tsmedizin, Humboldt University Berlin, Augustenburger Platz 1, D-13353, Berlin, Germany. Dr . Mary Petrou University College London, Institute of Women’s Health, Haemoglobinopathy Genetics Centre (Perinatal Centre), University College London Hospitals NHS Foundation Trust, London, UK. Dr . Martin Philpott Senior Research Fellow, Discipline of Nutrition, Faculty of Medical and Health Sciences, xiv on gene variation, genetic counseling, patenting of genes and of genetic tests, safety and quality management, and various ethical considerations and psychological issues per- taining to diagnostics. We feel that the inclusion of the lat- ter issues in this reference book have great relevance to our society and the aim is to assist in finding answers to some of the problematic questions that undoubtedly will arise, since application of molecular diagnostics may precipitate an important ethical crisis that physicians and the commu- nities they serve will be confronted with. The intended audience of this book is university post- graduate students from various life sciences disciplines, physicians, scientists in human molecular genetics and medicine, professionals working in diagnostic laboratories in academia or industry, academic institutions, hospital libraries, biotechnology and pharmaceutical companies. We believe that this book will be of help to decision- making advisors in medical insurance companies. In addi- tion, undergraduate medical and life science students will find very useful the description and explanation of recent modern techniques in life sciences. A major concern was to formulate the book contents in such a way that the notions described therein are clear and explained in a simple lan- guage and terminology. The numerous illustrations of the book are comprehensive and self-descriptive, and the glos- sary at the end of this book provides a brief explanation of the most commonly found terms. We expect that some points in this book can be further improved. We would welcome comments and criticism from the attentive readers, which will contribute to the improvement of the content of this book in its future edi- tions as well as helping to establish it as a reference publi- cation in the field of molecular diagnostics. Without the support and contributions of many peo- ple, the completion of this book would not be possible. We wish to thank the anonymous referees who supported our proposal and subsequently the people at Elsevier, in partic- ular Dr. Tessa Picknett and her department, whose interest in this title has encouraged us to proceed. We are also grateful to the editors, Drs. Claire Minto and Tari Paschall, and the senior production manager, Sarah Hajduk, at Elsevier, who helped us in a close collaboration to solve and overcome encountered difficulties. Their effi- ciency, pleasant manner, and patience added immensely to the smooth completion of this project from the very start. We also express our gratitude to all contributors for delivering outstanding compilations that summarize their experience and many years of hard work in their field of research. We are indebted to Julio Esperas who was respon- sible for the design and the cover of this book and to the copy editor, Adrienne Rebello, who has refined the final manuscript prior to going into production. We also thank our university colleagues, in particular Prof. Miel Ribbe from the Medical School of the University of Amsterdam, for discussions of the content of this book and ensuring its greatest possible relevance. We owe our thanks to the academic reviewers for their constructive criticisms on the chapters. We wish also to thank our families, from whom we have taken considerable amounts of time to dedicate to this work and whose patience and support have been conducive to the successful completion of this project. George P. Patrinos Wilhelm J. Ansorge Erasmus Medical Centre European Molecular Rotterdam, The Netherlands Biology Laboratory Heidelberg, Germany June 2005 Preface – First Edition xv Preface – Second Edition Four years after the publication of the first edition of Molecular Diagnostics , we are pleased to deliver the sec- ond edition of this textbook. Molecular diagnostics is a rap- idly evolving field, particularly after the deciphering of the human genome sequence. The need for a second edition of such a book derives not only from the recent discoveries and intellectual revolution in biomedical science but also from the rapid technological advancements applicable to low-cost and high-throughput molecular diagnostics, par- ticularly pertaining to nanotechnology. Over these last four years that this textbook has been available to the scientific community, it has been adopted as key reference in the field. This is clearly demonstrated from (a) the large number of copies sold worldwide in a relatively short time, (b) the various post-graduate and spe- cialist training courses on molecular diagnostics that have been used as a syllabus, (c) the adoption by universities as a textbook for related undergraduate courses and curricula, which also led to its translation in 2008, (d) the various positive reviews obtained not only from external reviewers, e.g. Doody’s and scientific journals, but also from fellow academics and students. The above have encouraged us and prompted Elsevier/Academic Press to move forward with the compilation of a second edition. As with the previous edition, our effort has been assisted by many internationally renowned experts in their field, who have kindly accepted our invitation to compile the 40 chapters of this book and share with us and our readers their expertise, experience, and results. The second edition kept the original structure of the first edition, which was one of its main innovative aspects. Each chapter contains an expert introduction to each sub- ject, next to few technical details and many applications for molecular genetic testing, which can be found in com- prehensive reference lists at the end of each chapter. The contents of this book are divided in two parts. The first part is dedicated to the battery of the most widely used molecular biology techniques. Their arrangement is in a more or less chronologic order of their development. In order to keep pace with the recent developments, few chap- ters from the first edition have been omitted, others have been merged while a large number of chapters pertaining to high-throughput molecular diagnostic approaches, e.g. oligonucleotide microarrays, next-generation sequencing, mass spectrometry, MLPA, etc., have been included. The remaining chapters have been updated compared to the pre- vious edition, to include not only technology innovations but also novel diagnostic applications. This resulted in the book being completely updated with over half of its con- tent being new compilations. As with the first edition, each of these chapters includes the principle and a brief descrip- tion of the technique, followed by examples from the area of expertise from the selected contributor. The second part attempts to integrate the previously analyzed technology to the different aspects of molecular diagnostics, such as identification of genetically modified organisms, stem cells, pharmacogenomics, modern foren- sic science, genetic quality of laboratory animals, molecu- lar microbiology, and preimplantation genetic diagnosis. Again, new chapters on areas where molecular genetic test- ing became relevant in the recent years, such as biodefense, victim identification in mass disasters, nutritional genom- ics (nutrigenomics), etc., have been included in this second edition. Finally, various everyday issues in a diagnostic laboratory, from locus-specific and national/ethnic muta- tion databases to gene patents and genetic counseling and related ethical and psychological issues to safety and qual- ity management, are discussed. As with the first edition, we feel that the inclusion of the latter issues in this reference book has still great relevance to our society. In addition, we made an effort to formulate the book contents such that the notions described therein are explained in a simple language and terminology for the book to be useful not only to experienced physicians and health care specialists and academics but also to undergrad- uate medical and life science students, and the numerous self-explanatory illustrations and glossary clearly contrib- ute to this end. Next, we tried to be consistent with the offi- cial gene and genetic variation nomenclature throughout the compilation. We are grateful to those colleagues who construc- tively criticized the first edition and identified deficiencies xvi that have been, hopefully, rectified in this second edition. However, we expect that some points in this book can still be further improved. Therefore, we would welcome com- ments and criticisms from attentive readers, which will contribute to improve the contents of this book even further in its future editions. We are also grateful to the editors, Tari Broderick, April Graham, and Janice Audet at Elsevier, who helped us in close collaboration to overcome encountered difficulties. We also express our gratitude to all contributors for delivering outstanding compilations that summarize their experience and many years of hard work in their field of research. We are indebted to Greg Harris who was responsible for cover design of this book and to the Elsevier production team (Alan Everett, Joe Howarth, Deena Burgess and Kim Lander) who refined the final manuscript. We owe our thanks to the academic reviewers for their constructive criticisms on the chapters and their positive evalution of our proposal for the second edition. Last , but not least, we wish to cordially thank our fami- lies for their patience and continuous support over the years, from whom we have taken a considerable amount of time to devote to this project. George P. Patrinos Wilhelm J. Ansorge University of Patras, Faculty of Health Sciences, Ecole Polytechnique Federal Lausanne, Department of Pharmacy Lausanne, Switzerland Patras, Greece Erasmus MC, Faculty of Medicine and Health Sciences, Department of Bioinformatics Rotterdam, The Netherlands September 2009 Preface – Second Edition 1 Molecular Diagnostics Copyright © 2010 Elsevier Ltd. All rights reserved. Molecular Diagnostics: Past, Present, and Future George P. Patrinos 1, 2 and Wilhelm J. Ansorge 3 1 Department of Pharmacy, School of Health Sciences, University of Patras, Patras, Greece; 2 Erasmus University Medical Center, Faculty of Medicine and Health Sciences, Department of Bioinformatics, Rotterdam, The Netherlands; 3 Ecole Polytechnique Federal Lausanne, EPFL, Lausanne, Switzerland Chapter 1 1.1 INTRODUCTION Molecular or nucleic acid-based diagnosis of human dis- orders is referred to as the detection of the various patho- genic mutations in DNA and/or RNA samples in order to facilitate detection, diagnosis, subclassification, prognosis, and monitoring response to therapy. Molecular diagnostics combines laboratory medicine with the knowledge and technology of molecular genetics and has been enormously revolutionized over the last decades, benefiting from the discoveries in the field of molecular biology (see Table 1.1 ). The identification and fine characterization of the genetic basis of the disease in question is vital for accurate pro- vision of diagnosis. Gene discovery provides invaluable insights into the mechanisms of disease, and gene-based markers allow physicians not only to assess disease pre- disposition but also to design and implement improved diagnostic methods. The latter is of great importance, as the plethora and variety of molecular defects demands the use of multiple rather than a single mutation detection plat- form. Molecular diagnostics is currently a clinical reality with its roots deep into the basic study of gene expression and function. 1.2 HISTORY OF MOLECULAR DIAGNOSTICS: INVENTING THE WHEEL In 1949, Pauling and his coworkers introduced the term molecular disease into the medical vocabulary, based on their discovery that a single amino acid change at the β -globin chain leads to sickle cell anemia, characterized mainly by recurrent episodes of acute pain due to vessel occlusion. In principle, their findings have set the founda- tions of molecular diagnostics, although the big revolution occurred many years later. At that time, when molecular biology was only hectically expanding, the provision of molecular diagnostic services was inconceivable and tech- nically not feasible. The first seeds of molecular diagnos- tics were provided in the early days of recombinant DNA technology, with many scientists from various disciplines working in concert. cDNA cloning and sequencing were at that time invaluable tools for providing the basic knowl- edge on the primary sequence of various genes. The latter provided a number of DNA probes, allowing the analysis via Southern blotting of genomic regions, leading to the concept and application of restriction fragment length poly- morphism (RFLP) to track a mutant allele from hetero- zygous parents to a high-risk pregnancy. In 1976, Kan and coworkers carried out, for the first time, prenatal diagno- sis of α -thalassemia, using hybridization on DNA isolated from fetal fibroblasts. Also, Kan and Dozy , in 1978, imple- mented RFLP analysis to pinpoint sickle cell alleles of African descent. This breakthrough provided the means of establishing similar diagnostic approaches for the charac- terization of other genetic diseases, such as phenylketonu- rea ( Woo et al. , 1983 ), cystic fibrosis ( Farrall et al. , 1986 ), and so on. At that time, however, a significant technical bottleneck had to be overcome. The identification of the disease caus- ing mutation was possible only through the construction of a genomic DNA library from the affected individual, in order first to clone the mutated allele and then determine its nucleotide sequence. Again, many human globin gene mutations were among the first to be identified through such approaches ( Busslinger et al. , 1981 ; Treisman et al. , 1983 ). In 1982, Orkin and his coworkers showed that a number of sequence variations were linked to specific β -globin gene mutations. These groups of RFLPs, termed haplotypes (both intergenic and intragenic), have provided a Molecular Diagnostics2 first-screening approach in order to detect a disease-causing mutation. Although this approach enabled researchers to predict which β -globin gene would contain a mutation, sig- nificantly facilitating mutation screening, no one was in the position to determine the exact nature of the disease-causing mutation, as many different β -globin gene mutations were linked to a specific haplotype in different populations (further information is available at http://globin.bx.psu. edu/hbvar ; Hardison et al. , 2002 ; Patrinos et al. , 2004 ; Giardine et al. , 2007 ). At the same time, in order to provide a shortcut to DNA sequencing, a number of exploratory methods for pin- pointing mutations in patients ’ DNA were developed. The first methods involved mismatch detection in DNA/DNA or RNA/DNA heteroduplexes ( Myers et al. , 1985a, b ) or dif- ferentiation of mismatched DNA heteroduplexes using gel electrophoresis, according to their melting profile ( Myers et al. , 1987 ). Using this laborious and time-consuming approach, a number of mutations or polymorphic sequence variations have been identified, which made possible the design of short synthetic oligonucleotides that were used as allele-specific probes onto genomic Southern blots. This experimental design was quickly implemented for the detection of β -thalassemia mutations ( Orkin et al. , 1983 ; Pirastu et al. , 1983 ). Despite the intense efforts from different laboratories worldwide, diagnosis of inherited diseases on the DNA level was still underdeveloped and therefore still not ready to be implemented in clinical laboratories for routine anal- ysis of patients due to the complexities, costs, and time requirements of the technology available. It was only after a few years that molecular diagnosis entered its golden era with the discovery of the most powerful molecular biology tool since cloning and sequencing, the polymerase chain reaction (PCR). 1.3 THE PCR REVOLUTION: GETTING MORE OUT OF LESS The discovery of PCR ( Saiki et al. , 1985 ; Mullis and Faloona, 1987 ) and its quick optimization, using a ther- mostable Taq DNA polymerase from Thermus aquaticus ( Saiki et al. , 1988 ) has greatly facilitated and in principle revolutionized molecular diagnostics. The most powerful feature of PCR is the large amount of copies of the target sequence generated by its exponential amplification (see Fig. 1.1 ), which allows the identification of a known muta- tion within a single day, rather than months. Also, PCR has markedly decreased or even diminished the need for radio- activity for routine molecular diagnosis. This has allowed molecular diagnostics to enter the clinical laboratory for the provision of genetic services, such as carrier or population screening for known mutations, prenatal diagnosis of inher- ited diseases, or in recent years, identification of unknown mutations, in close collaboration with research laboratories. Therefore, being moved to their proper environment, the clinical laboratory, molecular diagnostics could provide the services for which they have been initially conceived. The discovery of PCR has also provided the foundations for the design and development of many mutation detection schemes, based on amplified DNA. In general, PCR either is used for the generation of the DNA fragments to be ana- lyzed, or is part of the detection method. The first attempt was the use of restriction enzymes ( Saiki et al. , 1985 ) or oligonucleotide probes, immobilized onto membranes or in solution ( Saiki et al. , 1986 ) in order to detect the existing genetic variation, in particular the sickle cell disease-causing TABLE 1.1 The timeline of the principal discoveries in the field of molecular biology, which influenced the development of molecular diagnostics. Date Discovery 1949 Characterization of sickle cell anemia as a molecular disease 1953 Discovery of the DNA double helix 1958 Isolation of DNA polymerases 1960 First hybridization techniques 1969 In situ hybridization 1970 Discovery of restriction enzymes and reverse transcriptase 1975 Southern blotting 1977 DNA sequencing 1983 First synthesis of oligonucleotides 1985 Restriction fragment length polymorphism analysis 1985 Invention of PCR 1986 Development of fluorescent in situ hybridization (FISH) 1988 Discovery of the thermostable DNA polymerase – Optimization of PCR 1992 Conception of real-time PCR 1993 Discovery of structure-specific endonucleases for cleavage assays 1996 First application of DNA microarrays 2001 First draft versions of the human genome sequence 2001 Application of protein profiling in human diseases 2005 Introduction of the high-throughput next-generation sequencing technology Chapter | 1 Molecular Diagnostics: Past, Present, and Future 3 mutation. In the following years, an even larger number of mutation detection approaches have been developed and implemented. These techniques can be divided roughly into three categories, depending on the basis for discriminating the allelic variants: 1. Enzymatic-based methods . RFLP analysis was histori- cally the first widely used approach, exploiting the alterations in restriction enzyme sites, leading to the gain or loss of restriction events ( Saiki et al. , 1985 ). Subsequently, a number of enzymatic approaches for mutation detection have been conceived, based on the dependence of a secondary structure on the primary DNA sequence. These methods exploit the activity of resolvase enzymes T4 endonuclease VII, and, more recently, T7 endonuclease I to digest heteroduplex DNA formed by annealing wild-type and mutant DNA ( Mashal et al. , 1995 ). Digestion fragments indicate the presence and the position of any mutations. A variation of the theme involves the use of chemical agents for the same purpose ( Saleeba et al. , 1992 ; see also Chapter 3). Another enzymatic approach for mutation detection is the oligonucleotide ligation assay ( Landegren et al. , 1988 ) . In this technique, two oligonucleotides are hybridized to complementary DNA stretches at sites of possible mutations. The oligonucleotides ’ primers are designed such that the 3  end of the first primer is immediately adjacent to the 5  end of the second primer. Therefore, if the first primer matches completely with the target DNA, then the primers can be ligated by DNA ligase. On the other hand, if a mismatch occurs at the 3  end of the first primer, then no ligation products will be obtained. 2. Electrophoretic-based techniques . This category is characterized by a plethora of different approaches designed for screening of known or unknown muta- tions, based on the different electrophoretic mobility of the mutant alleles, under denaturing or non-denaturing conditions. Single strand conformation polymorphism (SSCP) and heteroduplex (HDA) analyses ( Orita et al. , 1989 ; see Chapter 4) were among the first meth- ods designed to detect molecular defects in genomic loci. In combination with capillary electrophoresis (see Chapter 5) , SSCP and HDA analysis now provide an excellent, simple, and rapid mutation detection platform with low operation costs and, most interestingly, the potential of easily being automated, thus allowing for high-throughput analysis of patients ’ DNA. Similarly, denaturing and temperature gradient gel electrophoresis (DGGE and TGGE, respectively) can be used equally well for mutation detection (see Chapter 6) . In this case, electrophoretic mobility differences between a wild-type and mutant allele can be ‘ ‘ visualized ’ ’ in a gradient of denaturing agents, such as urea and formamide, or of increasing temperature. Finally, an increasingly used mutation detection technique is the two-dimensional gene scanning , based on two- dimensional electrophoretic separation of amplified DNA fragments, according to their size and base pair sequence. The latter involves DGGE, following the size separation step. 3. Solid phase-based techniques . This set of techniques consists of the basis for most of the present-day muta- tion detection technologies, since they have the extra advantage of being easily automated and hence are highly recommended for high-throughput mutation detection or screening. A fast, accurate, and convenient method for the detection of known mutations is reverse dot-blot, initially developed by Saiki and coworkers (1989) and implemented for the detection of β -thalassemia mutations. The essence of this method is the utilization of oligonucleotides, bound to a mem- brane, as hybridization targets for amplified DNA. Some of this technique’s advantages is that one mem- brane strip can be used to detect many different known mutations in a single individual (a one strip/one patient type of assay), the potential of automation, and the ease of interpretation of the results, using a classical avidin- biotin system. However, this technique cannot be used FIGURE 1.1 The PCR principle. Thick and thin black lines correspond to the target sequence and genomic DNA, respectively; gray boxes cor- respond to the oligonucleotide primers, and the correct size PCR products are included in the white ellipses. Dashed lined arrows depict the elonga- tion of the template strand. Molecular Diagnostics6 1.5 FUTURE PERSPECTIVES: WHAT LIES BEYOND As an intrinsic part of DNA technology, molecular diag- nostics are rooted in the April 1953 discovery of the DNA double helix. Today, it is clear that they embody a set of notable technological advances allowing for thousands of diagnostic reactions to be performed at once and for a range of mutations to be simultaneously detected. The rea- sons for this dramatic increase are two-fold. First of all, the elucidation of the human genomic sequence, as well as that of other species such as bacterial or viral pathogens, has led to an increased number of diagnostically relevant targets. Second, the molecular diagnostic testing volume is rapidly increasing. This is the consequence of a better understanding of the basis of inherited diseases, therefore allowing molecular diagnostics to play a key role in patient or disease management. Presently , a great number of blood, hair, semen, and tissue samples are analyzed annually worldwide in both public and private laboratories, and the number of genetic tests available is steadily increased year by year. Taking these premises into account, we can presume that it is only a matter of time before molecular diagnostic laboratories become indispensable in laboratory medicine. In the post- genomic era, genetic information will have to be examined in multiple health care situations throughout people’s lives. Currently, newborns can be screened for phenylketonurea and other treatable genetic diseases ( Yang et al. , 2001 ). It is also possible that in the not-so-distant future, children at high risk from coronary artery disease will be identified and treated to prevent changes in their vascular walls dur- ing adulthood. Similarly, parents will have the option of being informed about their carrier status for many reces- sive diseases before they decide to start a family. Although not widely accepted, this initiative has already started to be implemented in Cyprus, where a couple at risk for tha- lassemia syndrome have been advised to undergo a genetic test for thalassemia mutations before their marriage (see also Chapter 37) . Also, for middle-aged and older popula- tions, scientists will be able to determine risk profiles for various late-onset diseases, preferably before the appear- ance of symptoms, which at least could be partly prevented through dietary or pharmaceutical interventions. In the near future, the monitoring of individual drug response profiles throughout life, using genetic testing for the identifica- tion of their individual DNA signature, will be part of the standard medical practice. Soon, genetic testing will com- prise a wide spectrum of different analyses with a host of consequences for individuals and their families, which is worth emphasizing when explaining molecular diagnos- tics to the public (see also Chapter 38) . All these issues are discussed in detail next. However, and in order to be more realistic, many of these expectations still are based on promises, though quite optimistic ones. Thus, some of the new perspectives of the field could be a decade away, and several challenges remain to be realized. 1.5.1 Commercializing Molecular Diagnostics Currently , clinical molecular genetics is part of mainstream health care worldwide. Almost all clinical laboratories have a molecular diagnostic unit or department. Although in recent years the notion of molecular diagnostics has increasingly gained interest, genetic tests are still not gener- ally used for population screening, but rather for diagnosis, carrier screening, and prenatal diagnosis, and only on a lim- ited basis. Therefore, and in order to make molecular diag- nostics widely available, several obstacles and issues need to be taken into consideration and resolved in the coming years. The first important issue is the choice of the mutation detection platform. Despite the fact that there are over 50 different mutation detection and screening methods, there is no single platform or methodology that prevails for genetic testing. Genotyping can be done using different approaches, such as filters, gels, microarrays, microtiter plates; different amplification-based technologies; different separation techniques, such as blotting, capillary electro- phoresis, microarrays, mass spectroscopy; and finally dif- ferent means for labeling, such as radioactive, fluorescent, chemiluminescent, or enzymatic substances. The variety of detection approaches makes it not only difficult but also challenging to determine which one is better suited for a laboratory setting. Generally speaking, DNA sequenc- ing is the gold standard for the identification of causative or non-DNA sequence variations, particularly with the advent of the next-generation sequencing technologies (see also Chapter 24) . The initial investment costs and the expected test volume are some of the factors that need to be taken into consideration prior to choosing the detection technique. Related issues are also the costs of the hard- ware and software, testing reagents, and kits. The latter is of great importance, since the fact that most of the diag- nostic laboratories today are running ‘ ‘ home-brew ’ ’ assays – for example, not using well-standardized genetic testing kits due to cost barriers, which brings to surface the issue of quality control of the reagents (see Chapter 40) and of safety (see Chapter 39) . Currently, there are several clini- cal and technical recommendations for genetic testing for monogenic disorders that have been issued by several organizations (see Table 1.2 ). Another very important issue is training the personnel of a molecular diagnostic laboratory, reflecting in the quality and the correct interpretation of the results. Continuous education of the personnel of the diagnostic laboratory is crucial for the accuracy of the results provided (see also Chapter 40). Many times, such as in the case of prenatal or pre-implantation Chapter | 1 Molecular Diagnostics: Past, Present, and Future 7 diagnosis, irrevocable decisions need to be made, most of the time based on a simple test result. In the past decade, there has been a significant reduction in the number of incorrect genotypes diagnosed, as a result of continuous training and proficiency testing schemes ( http://www.eurogentest.org ). In the USA, there is a voluntary biannual proficiency test- ing for molecular diagnostic laboratories, while in Europe, the EuroGenTest European Network of Excellence ( http:// www.eurogentest.org ) has been founded to promote quality in molecular genetic testing through the provision of exter- nal quality assessment (proficiency testing schemes) and the organization of best practice meetings and publication of guidelines. It is generally true that many geneticists and non- geneticist physicians would benefit from continuous education regarding the appropriate use of molecular diagnostic tests, which is necessary to evaluate the method pre-analytically and to interpret results. The legal considerations and the ethical concerns are also hurdles that need to be overcome in the coming years. One issue is reimbursing of the diagnosis costs. At present, there are no insurance companies that reimburse the costs for molecular testing to the people insured; the necessary regulatory and legal framework remains to be established. ‘ ‘ Legalizing ’ ’ molecular testing, by the adoption of the rele- vant regulations, would probably result in an increase of the test volume and at the same time it can pose an immense barrier to uncontrolled genetic testing. Similarly, the need to obtain an informed consent from the patient to be ana- lyzed is also of great importance and should be encouraged and facilitated by the diagnostic laboratory. On the other hand, the issue of intellectual proper- ties hampers the wide commercialization of molecular diagnostics. Almost all the clinically relevant genes have been now patented and the terms that the patent holders offer vary considerably (see Chapter 36) . Among the dif- ficulties that this issue imposes is the limiting choice of mutation detection platforms, the large loyalties for rea- gent use, and the exclusive sublicenses that many com- panies grant to clinical laboratories, leading eventually to monopolies. Since one of the biggest challenges that the clinical laboratory is facing is patent and regulatory com- pliance, partnerships and collaborations may be envisaged in order to take the technology licenses to the diagnostic laboratory that will subsequently develop, standardize, and distribute the assays. These will partly alleviate some of the intellectual properties issues. Finally, the issue of the medical genetics specialty is more urgent than ever. In the USA, medical genetics has been formally recognized as a medical specialty only within the past 15 years, and in Europe, medical genetics only recently has been for- mally recognized as a specialty ( http://www.eshg.org ). The implementation of this decision is still facing sub- stantial difficulties ( http://www.eshg.org/geneticseurope. htm ), which will probably take years to bypass. With the completion of the Human Genome Project, genetics has become the driving force in medical research and is now poised for integration into medical practice. An increase in the medical genetics workforce, including geneticists and genetic counselors, will be necessary in the coming years. After all, the Human Genome Project has made informa- tion of inestimable diagnostic and therapeutic importance available and therefore the medical profession now has the obligation to rise to both the opportunities and challenges that this wealth of genetic information presents. TABLE 1.2 Indicative clinical and technical recommendations for genetic testing for monogenic disorders. ACMG: American College of Medical Genetics, ASHG: American Society of Human Genetics. Disease/syndrome Gene References Alzheimer’s disease ApoE ACMG (1995) Canavan disease ASPA ACMG (1998) Cystic fibrosis CFTR Dequeker et al. (2000) , Grody et al. (2001a) Thrombophilia Factor V Leiden Grody et al. (2001b) Fragile X syndrome FMR1 Maddalena et al. (2001) Prader – Willi/Angelman syndrome 15q11-q13 ASHG/ACMG (1996) Multiple endocrine neoplasia MEN1/MEN2 Brandi et al. (2001) Tuberous sclerosis TSC1/TSC2 Roach et al. (1999) Breast cancer BRCA1 Sorscher and Levonian (1997) Molecular Diagnostics8 1.5.2 Personalized Medicine The term ‘ ‘ personalized medicine ’ ’ refers to the practice of medicine where patients receive the most appropri- ate medical treatment, fitting dosage, and combination of drugs based on their genetic background. Some of the rea- sons for many types of adverse drug reactions are already known and often related to polymorphic gene alleles of drug metabolizing enzymes ( Nebert and Menon, 2001 ; Risch et al. , 2002 ). The application of high-throughput genotyping tools for the identification and screening of sin- gle nucleotide polymorphisms (SNPs) eventually can lead to the determination of the unique molecular signature of an individual in a relatively short period of time. This way, individual drug responses can be predicted from predeter- mined genetic variances correlated with a drug effect. In other words, this will allow the physician to provide the patient with a selective drug prescription (see Chapter 22) . A handful of pharmaceutical companies are developing a precise haplotyping scheme to identify individuals/patients who will derive optimal benefit from drugs currently under development. Clinicians will facilitate this effort by import- ing clinical data into this haplotyping system for a complete patient analysis and drug evaluation. In addition to these efforts, there is a growing need to incorporate this increas- ingly complex body of knowledge to standard medical practice. Incorporating pharmacogenomics-related courses in the standard curriculum of medical schools potentially can ensure that the forthcoming generation of clinicians and researchers will be familiar with the latest develop- ments in that field and will be capable of providing patients with the expected benefits of personalized medicine. Similarly , nutrigenomics (or nutritional genomics) inves- tigates the interactions between nutrition and an individual’s genome, and the consequent downstream effects on their phenotype with the aim of providing tailored nutritional advice or developing specialist food products (see Chapter 23) . In other words, nutrigenomics recognizes that specific dietary advice that can be beneficial for one individual may be inappropriate, or actually harmful, to another. Although comparable to pharmacogenomics, nutrigenomics is still considered as an emerging science contrary to pharma- cogenomics, which is considered to have ‘ ‘ come of age ’ ’ ( Allison, 2008 ). However , there are growing concerns regarding the eth- ical aspects of personalized medicine. First of all, equality in medical care needs to be ensured, when genetics fore- tell clinicians which patients would be less likely to benefit from a particular drug treatment. Second, it will become increasingly vital to devise operational tools for the preven- tion of stigmatization and discrimination of different popu- lations, in particular on ethnic grounds (van Ommen, 2002 ), and therefore every precaution should be taken to elimi- nate all lingering prejudice and bias associated with the study of human genetic variation. Other dilemmas include the right to deny an available treatment from specific patient populations according to genetic-derived indications, as currently is the case with prenatal diagnosis (see also Chapter 37) . Appropriate guidelines will be crucially needed for the successful implementation of pharmacogenomics into clinical practice. 1.5.3 Personal Genomics The ultimate goal in health care over the next decades will be the efficient integration of molecular diagnostics with therapeutics. With the advent of next generation sequenc- ing in 2005 ( Margulies et al. , 2005 ) and the avalanche of developments in this field since then (see Chapter 24) , experts believe that reasonably soon, people will be able to have their own genomes sequenced for under $1,000. This is going to involve sequencing technology that is much cheaper and faster than today’s machines and several efforts are currently under way, often encouraged by major funding bodies (e.g. the European Commission-funded READNA consortium; http://www.cng.fr/READNA ). When that point is reached, this can ultimately be translated in a patient being able to carry a smart card, like an ordinary credit card, providing secure access to his or her genetic information. So far, the entire DNA sequence of a handful of individuals has been sequenced, such as Craig Venter ( Levy et al. , 2007 ), Jim Watson ( Wheeler et al. , 2008 ), and so on, while ‘ ‘ the 1000 genomes ’ ’ project is an ambi- tious venture that involves sequencing the genomes of approximately 1,200 people from around the world, with the overall goal ‘ ‘ . . . to create the most detailed and medi- cally useful picture to date of human genetic variation ’ ’ ( http://www.1000genomes.org ). In the future, a person may appear at the clinic for treatment, ‘ ‘ carrying ’ ’ its entire genome at hand or, alternatively, nanotechnology could even- tually enable DNA analysis with a portable DNA sequencing device. Even though the expectations are high and compa- nies are currently using these new technologies to provide information to individuals to predict health and disease outcome, even behavioral traits, it is generally premature to make promises for clinically useful information from genomic analyses. Next to that, there is an inherent dan- ger of overestimating the usefulness of the various per- sonalized genomic tests that can be ordered directly by consumers. Unlike other genetic analyses, these tests pro- vide sheer amounts of genetic information, but their diag- nostic or prognostic value remains uncertain because of (a) the lack of information about the influence of environmen- tal and other factors, and (b) the weak association for the vast majority of genetic loci with disease. In a carefully conducted study by Janssens and coworkers (2008) , the scientific evidence supporting gene – disease associations for genes included in genomic tests offered commercially Chapter | 1 Molecular Diagnostics: Past, Present, and Future 11 Risch , N. , Burchard , E. , Ziv , E. , and Tang , H. ( 2002 ) . Categorization of humans in biomedical research: genes, race and disease . Genome Biol. 3 , 1 – 12 . Roach , E.S. , DiMario , F.J. , Kandt , R.S. , and Northrup , H. ( 1999 ) . Tuberous Sclerosis Consensus Conference: recommendations for diagnostic evaluation. National Tuberous Sclerosis Association . J. Child Neurol. 14 , 401 – 407 . Robinson , W.H. , DiGennaro , C. , Hueber , W. , Haab , B.B. , Kamachi , M. , Dean , E.J. , Fournel , S. , Fong , D. , Genovese , M.C. , de Vegvar , H.E. , Skriner , K. , Hirschberg , D.L. , Morris , R.I. , Muller , S. , Pruijn , G.J. , van Venrooij , W.J. , Smolen , J.S. , Brown , P.O. , Steinman , L. , and Utz , P.J. ( 2002 ) . Autoantigen microarrays for multiplex characterization of autoantibody responses . Nat. Med. 8 , 295 – 301 . Ronaghi , M. , Uhlen , M. , and Nyren , P. ( 1998 ) . A sequencing method based on real-time pyrophosphate . Science 281 , 363 – 365 . Saiki , R.K. , Scharf , S. , Faloona , F. , Mullis , K.B. , Horn , G.T. , Erlich , H.A. , and Arnheim , N. ( 1985 ) . Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia . Science 230 , 1350 – 1354 . Saiki , R.K. , Bugawan , T.L. , Horn , G.T. , Mullis , K.B. , and Erlich , H.A. ( 1986 ) . Analysis of enzymatically amplified beta-globin and HLA- DQ alpha DNA with allele-specific oligonucleotide probes . Nature 324 , 163 – 166 . Saiki , R.K. , Gelfand , D.H. , Stoffel , S. , Scharf , S.J. , Higuchi , R. , Horn , G.T. , Mullis , K.B. , and Erlich , H.A. ( 1988 ) . Primer directed enzymatic amplification of DNA with a thermostable DNA polymerase . Science 239 , 487 – 491 . Saiki , R.K. , Walsh , P.S. , Levenson , C.H. , and Erlich , H.A. ( 1989 ) . Genetic analysis of amplified DNA with immobilized sequence-specific oligo- nucleotide probes . Proc. Natl. Acad. Sci. USA 86 , 6230 – 6234 . Saleeba , J.A. , Ramus , S.J. , and Cotton , R.G. ( 1992 ) . Complete mutation detection using unlabeled chemical cleavage . Hum. Mutat. 1 , 63 – 69 . Sorscher , S. , and Levonian , P. ( 1997 ) . BCRA 1 testing guidelines for high- risk patients . J. Clin. Oncol. 15 , 1711 . Treisman , R. , Orkin , S.H. , and Maniatis , T. ( 1983 ) . Specific transcrip- tion and RNA splicing defects in five cloned beta-thalassaemia genes . Nature 302 , 591 – 596 . van Ommen , G.J. ( 2002 ) . The Human Genome Project and the future of diagnostics, treatment and prevention . J. Inherit. Metab. Dis. 25 , 183 – 188 . Venter , J.C. , Adams , M.D. , Myers , E.W. , Li , P.W. , Mural , R.J. , Sutton , G.G. , Smith , H.O. , Yandell , M. , Evans , C.A. , Holt , R.A. , Gocayne , J.D. , Amanatides , P. , Ballew , R.M. , Huson , D.H. , Wortman , J.R. , Zhang , Q. , Kodira , C.D. , Zheng , X.H. , Chen , L. , Skupski , M. , Subramanian , G. , Thomas , P.D. , Zhang , J. , Gabor Miklos , G.L. , Nelson , C. , Broder , S. , Clark , A.G. , Nadeau , J. , McKusick , V.A. , Zinder , N. , Levine , A.J. , Roberts , R.J. , Simon , M. , Slayman , C. , Hunkapiller , M. , Bolanos , R. , Delcher , A. , Dew , I. , Fasulo , D. , Flanigan , M. , Florea , L. , Halpern , A. , Hannenhalli , S. , Kravitz , S. , Levy , S. , Mobarry , C. , Reinert , K. , Remington , K. , Abu-Threideh , J. , Beasley , E. , Biddick , K. , Bonazzi , V. , Brandon , R. , Cargill , M. , Chandramouliswaran , I. , Charlab , R. , Chaturvedi , K. , Deng , Z. , Di Francesco , V. , Dunn , P. , Eilbeck , K. , Evangelista , C. , Gabrielian , A.E. , Gan , W. , Ge , W. , Gong , F. , Gu , Z. , Guan , P. , Heiman , T.J. , Higgins , M.E. , Ji , R.R. , Ke , Z. , Ketchum , K.A. , Lai , Z. , Lei , Y. , Li , Z. , Li , J. , Liang , Y. , Lin , X. , Lu , F. , Merkulov , G.V. , Milshina , N. , Moore , H.M. , Naik , A.K. , Narayan , V.A. , Neelam , B. , Nusskern , D. , Rusch , D.B. , Salzberg , S. , Shao , W. , Shue , B. , Sun , J. , Wang , Z. , Wang , A. , Wang , X. , Wang , J. , Wei , M. , Wides , R. , Xiao , C. , Yan , C. , Yao , A. , Ye , J. , Zhan , M. , Zhang , W. , Zhang , H. , Zhao , Q. , Zheng , L. , Zhong , F. , Zhong , W. , Zhu , S. , Zhao , S. , Gilbert , D. , Baumhueter , S. , Spier , G. , Carter , C. , Cravchik , A. , Woodage , T. , Ali , F. , An , H. , Awe , A. , Baldwin , D. , Baden , H. , Barnstead , M. , Barrow , I. , Beeson , K. , Busam , D. , Carver , A. , Center , A. , Cheng , M.L. , Curry , L. , Danaher , S. , Davenport , L. , Desilets , R. , Dietz , S. , Dodson , K. , Doup , L. , Ferriera , S. , Garg , N. , Gluecksmann , A. , Hart , B. , Haynes , J. , Haynes , C. , Heiner , C. , Hladun , S. , Hostin , D. , Houck , J. , Howland , T. , Ibegwam , C. , Johnson , J. , Kalush , F. , Kline , L. , Koduru , S. , Love , A. , Mann , F. , May , D. , McCawley , S. , McIntosh , T. , McMullen , I. , Moy , M. , Moy , L. , Murphy , B. , Nelson , K. , Pfannkoch , C. , Pratts , E. , Puri , V. , Qureshi , H. , Reardon , M. , Rodriguez , R. , Rogers , Y.H. , Romblad , D. , Ruhfel , B. , Scott , R. , Sitter , C. , Smallwood , M. , Stewart , E. , Strong , R. , Suh , E. , Thomas , R. , Tint , N.N. , Tse , S. , Vech , C. , Wang , G. , Wetter , J. , Williams , S. , Williams , M. , Windsor , S. , Winn-Deen , E. , Wolfe , K. , Zaveri , J. , Zaveri , K. , Abril , J.F. , Guigo , R. , Campbell , M.J. , Sjolander , K.V. , Karlak , B. , Kejariwal , A. , Mi , H. , Lazareva , B. , Hatton , T. , Narechania , A. , Diemer , K. , Muruganujan , A. , Guo , N. , Sato , S. , Bafna , V. , Istrail , S. , Lippert , R. , Schwartz , R. , Walenz , B. , Yooseph , S. , Allen , D. , Basu , A. , Baxendale , J. , Blick , L. , Caminha , M. , Carnes-Stine , J. , Caulk , P. , Chiang , Y.H. , Coyne , M. , Dahlke , C. , Mays , A. , Dombroski , M. , Donnelly , M. , Ely , D. , Esparham , S. , Fosler , C. , Gire , H. , Glanowski , S. , Glasser , K. , Glodek , A. , Gorokhov , M. , Graham , K. , Gropman , B. , Harris , M. , Heil , J. , Henderson , S. , Hoover , J. , Jennings , D. , Jordan , C. , Jordan , J. , Kasha , J. , Kagan , L. , Kraft , C. , Levitsky , A. , Lewis , M. , Liu , X. , Lopez , J. , Ma , D. , Majoros , W. , McDaniel , J. , Murphy , S. , Newman , M. , Nguyen , T. , Nguyen , N. , Nodell , M. , Pan , S. , Peck , J. , Peterson , M. , Rowe , W. , Sanders , R. , Scott , J. , Simpson , M. , Smith , T. , Sprague , A. , Stockwell , T. , Turner , R. , Venter , E. , Wang , M. , Wen , M. , Wu , D. , Wu , M. , Xia , A. , Zandieh , A. , and Zhu , X. ( 2001 ) . The sequence of the human genome . Science 291 , 1304 – 1351 . Wen , W.H. , Bernstein , L. , Lescallett , J. , Beazer-Barclay , Y. , Sullivan- Halley , J. , White , M. , and Press , M.F. ( 2000 ) . Comparison of TP53 mutations identified by oligonucleotide microarray and conventional DNA sequence analysis . Cancer Res. 60 , 2716 – 2722 . Wheeler , D.A. , Srinivasan , M. , Egholm , M. , Shen , Y. , Chen , L. , McGuire , A. , He , W. , Chen , Y.J. , Makhijani , V. , Roth , G.T. , Gomes , X. , Tartaro , K. , Niazi , F. , Turcotte , C.L. , Irzyk , G.P. , Lupski , J.R. , Chinault , C. , Song , X.Z. , Liu , Y. , Yuan , Y. , Nazareth , L. , Qin , X. , Muzny , D.M. , Margulies , M. , Weinstock , G.M. , Gibbs , R.A. , and Rothberg , J.M. ( 2008 ) . The complete genome of an individual by massively parallel DNA sequencing . Nature 452 , 872 – 876 . Woo , S.L. , Lidsky , A.S. , Guttler , F. , Chandra , T. , and Robson , K.J. ( 1983 ) . Cloned human phenylalanine hydroxylase gene allows prenatal diag- nosis and carrier detection of classical phenylketonuria . Nature 306 , 151 – 155 . Xiao , W. , and Oefner , P.J. ( 2001 ) . Denaturing high-performance liquid chromatography: a review . Hum. Mutat. 17 , 439 – 474 . Yang , Y. , Drummond-Borg , M. , and Garcia-Heras , J. ( 2001 ) . Molecular analysis of phenylketonuria (PKU) in newborns from Texas . Hum. Mutat. 17 , 523 . This page intentionally left blank ( Section |) — Molecular Diagnostic Technology Molecular Diagnostics16 activity, and therefore cannot correct mismatches at the 3  terminus of the primer. As such, complementary base- pairing at the 3  end of the primer is required for efficient amplification by Taq DNA polymerase and is a strong determining factor of template specificity. Amplification of the normal allele, and not that of the mutant, is accom- plished using a primer that is complementary to the normal allele and has a mismatch between the 3  residue and the mutant allele. Conversely, only the mutant will be ampli- fied if the 3  residue of the primer is complementary to the mutant allele and not the normal allele. The specificity or discriminating power of the 3  terminal nucleotide can be enhanced further by incorporating an additional mismatch positioned near the 3  nucleotide ( Newton et al. , 1989 ). The basic concept is illustrated in Fig. 2.1 . Various studies have attempted to quantify the inhibi- tory effect of different 3  mismatches on PCR amplifica- tion ( Kwok et al. , 1990 ; Sarkar et al. , 1990 ; Huang et al. , 1992 ; Ayyadevara et al. , 2000 ). Although some trends have emerged, the results were remarkably discordant. Sarkar and coworkers (1990) concluded that PCR is inhib- ited by mismatches between the template and the 3  or 3  penultimate nucleotide of the primer. Under relatively relaxed stringency conditions for primer-template anneal- ing, Kwok and coworkers (1990) demonstrated a 20-fold reduction in amplification efficiency with A:A (primer- template) mismatches, 100-fold reductions with A:G, G: A, and C:C mismatches, and little or no reduction with any other mismatches. Under higher stringency condi- tions, Huang and coworkers (1992) showed some degree of inhibition with every combination of 3  mismatch. The weakest inhibition, about 100-fold reduction, was associ- ated with C:T mismatches. There was approximately 103- fold reduction with A:C, C:A, G:T, and T:G mismatches; 103- to 104-fold reduction with T:C and T:T mismatches; and at least 106-fold reduction with A:A, G:A, A:G, G:G, and C:C mismatches. Ayyadevara and coworkers (2000) systematically varied both the 3  terminal and penultimate nucleotides of primers, all under relatively high stringency conditions. Their study indicated that primers ending with 3  A are moderately inferior to those ending in other nucleotides. Allele-specific amplification had 40- to 100- fold reduction when the mismatched primer had T, G, or C at the 3  terminal position. They also concluded that the penultimate 3  nucleotide plays a minor role in mismatch discrimination, and that amplification efficiency is reduced when A (and to a lesser extent T) occupies the penultimate 3  position. The design and optimization of PCR-ARMS proto- cols is primarily a function of the target sequence and the nucleotide differences that define the alleles. In addition to mismatches between the 3  terminal base of the primer and the target, single mismatches should be incorporated at several positions from the 3  terminus. Apart from the the- oretical considerations relating to the 3  terminal position of the allele-specific primers, the design and optimization of PCR-ARMS primers follows the same considerations used for any other type of PCR. Primers are chosen to have comparable theoretical melting temperatures ( T m ). Primer lengths are generally 20 nucleotides or longer, although the length is less important than the T m . Primers should not have self-complementary sequences of 4 nucleotides or more, nor should they have more than 4 nucleotide comple- mentarity between their 3  ends. As with any PCR-based strategy, false negative results could result from the presence of sequence variations that negatively impact on the primer annealing and/or amplification. This potential problem can be overcome by targeting the opposite strand for amplification, or by incorporating a degenerate nucleotide into the primer. For single mutation ARMS, the PCR conditions can be estab- lished by titrating the MgCl 2 concentration and/or primer concentrations, at constant annealing temperature. For multiplex ARMS, the first step is to optimize the PCR con- ditions for sensitive and specific detection of each allele. The objective is to define the PCR cycling parameters and MgCl 2 concentration under which all of the alleles will be amplified in an efficient and specific manner. It may be necessary to redesign one or more primer pair to achieve allele-specific amplification under one set of PCR condi- tions. Once the PCR parameters have been established, the primer pairs can be combined to evaluate the perform- ance of the multiplex ARMS assay. Primer concentrations should be adjusted such that each of the alleles is ampli- fied to a comparable degree. The specificity of the ARMS assay should be evaluated using samples from normal con- trols and known carriers of the mutations. Specificity can also be tested using serial dilutions of mutant DNA mixed with normal DNA (e.g. mutant:normal  1:1, 1:2, 1:4, 1:8, etc.). Uniform and specific amplification of each allele may require further manipulation of the cycling parameters or the concentration of one or more reagents (primers, Taq polymerase, MgCl 2 ). FIGURE 2.1 Schematic of PCR-ARMS. Schematic representation of a PCR-ARMS assay for the detection of a single-base mutation (under- lined). The 3  terminal nucleotide of the ARMS primer is complementary to the mutant allele. The ARMS primer has an additional mismatch posi- tioned three bases from the 3  terminal nucleotide (not shown). Chapter | 2 Allele-Specific Mutation Detection 17 2.2.2 Single and Multiplex PCR-ARMS A common application of PCR-ARMS is the detection of individual point mutations in DNA. Primers are designed that will preferentially amplify the mutant allele, while being refractory to amplification of the normal allele. Included in the reaction mix is a second set of primers that are specific for a heterologous locus that serves as a posi- tive control for PCR amplification. Conventional agarose or polyacrylamide gel electrophoresis systems are used to resolve the control amplicon from the mutant amplicon. Since the efficiency of amplification is inversely propor- tional to the length of the amplicon, the control amplicon should be larger or close in size to the mutant amplicon. Figure 2.2 illustrates the use of this approach to detect the most common DHCR7 gene mutation associated with the Smith – Lemli – Opitz syndrome ( Nowaczyk et al. , 2001 ). This assay distinguishes between samples that are posi- tive for the IVS 8-1 G  C mutation (heterozygous or homozygous) and those that are negative for the mutation. This is sufficient for applications where it is not necessary to distinguish between heterozygotes and homozygotes (e.g. mutation analysis of unaffected carriers of a recessive disorder). For other applications, such as mutation analysis for individuals affected with a recessive disorder, it may be desirable to genotype individuals who are positive for the mutation. This can be accomplished by screening all posi- tive samples with a second ARMS assay that is specific for the normal allele and refractory for the mutant allele. Heterozygotes will be positive for both ARMS assays, whereas homozygotes will be positive for only the mutant ARMS assay. The specificity of PCR-ARMS is such that pools of samples can be screened to identify rare carriers of specific mutations. This approach is capable of detecting a single positive sample in pools of 30 or more samples. This eliminates the need to test large numbers of samples indi- vidually to establish population frequencies for individual mutant alleles ( Nowaczyk et al. , 2001 ; Waye et al. , 2002 ). Multiplex PCR-ARMS assays can easily be developed for single-tube detection of four to six different mutations ( Old et al. , 1990 ). Multiplexing PCR-ARMS assays is often necessary, since many genetic disorders are characterized by a small number of common mutations that account for a significant proportion, and in some instances, the major- ity of mutant alleles represented in a given population (see also Chapter 25) . Once the spectrum of mutations and the frequencies of individual alleles have been established, PCR-ARMS can be used to simultaneously screen for the most common mutations. Figure 2.3 shows two multiplex PCR-ARMS panels that detect 11 common DHCR7 gene mutations. Collectively, these mutations account for more than 85% of the mutant alleles detected in North American Smith-Lemli-Opitz syndrome patients. 2.2.3 Genotyping with PCR-ARMS PCR -ARMS can be used to determine genotypes for indi- vidual mutations or SNPs, using two separate ARMS assays: one specific for the mutant allele and the other spe- cific for the normal allele. Alternatively, PCR-ARMS sys- tems have been developed for single-tube genotyping of mutations or SNPs. The simplest system involves bidirec- tional amplification, with the normal allele amplified using one strand as the template and the mutant allele amplified off the complementary strand ( Waterfall and Cobb, 2001 ; Ye et al. , 2001 ). The primers are designed such that the lengths of the amplicons can easily be resolved by con- ventional gel electrophoresis. This strategy has a built-in FIGURE 2.2 PCR-ARMS detection of a single DHCR7 mutation. A. Schematic representation of the boundary between intervening sequence 8 and exon 8 of the DHCR7 gene showing the position of a common mutation causing Smith – Lemli – Opitz syndrome. The mutation alters the canonical splice acceptor sequence (AG  AC) and can be detected using an ARMS primer specific for the mutant allele. B. PCR-ARMS detection of the IVS 8-1 G Æ C mutation (underlined). The 3  terminal nucleotide of the ARMS primer is complementary to the mutant allele, and an addi- tional mismatch is incorporated three bases from the 3  nucleotide (not shown). C. Analysis of PCR-ARMS products by non-denaturing poly- acrylamide gel electrophoresis, visualized by ethidium bromide staining and UV fluorescence. The 190 bp fragment is specific for the IVS 8-1 G  C mutant allele. The 429 bp fragment corresponds to a region of the HFE gene that serves as a positive internal control for PCR amplification. Lane 1: IVS 8-1 G  C heterozygote; lane 2: normal control; lane 3: 1:1 mixture of carrier:normal DNA; lanes 4 – 13: 1:2n mixture (N  1 – 10) of carrier:normal DNA, respectively (1:2 to 1:1024). Molecular Diagnostics18 positive control resulting from amplification between the outermost primers of the normal and mutant amplicons. Figure 2.4 shows the application of PCR-ARMS to geno- type samples for the most common mutation associated with hereditary hemochromatosis ( HFE p.C282Y). PCR -ARMS can be also used to establish haplotypes of individuals in the absence of samples from relatives. This is particularly useful for haplotyping SNPs that are located within distances that are amenable to PCR amplification. Consider the case of adjacent bi-allelic SNPs, where the alleles are designated Aa and Bb. PCR-ARMS using the four possible combinations of ARMS primers specific for the SNP A and SNP B alleles (AB, Ab, aB, ab) can be used to establish the haplotype ( Eitan and Kashi, 2002 ). 2.2.4 Advantages and Limitations PCR -ARMS assays are ideally suited for many molecu- lar diagnostic applications, particularly those requiring detection of relatively small numbers of point mutations and having low-to-moderate throughputs. The primary advantage of PCR-ARMS is the ease with which multi- plex assays can be developed, validated, and implemented. Moreover, the tests are non-radioactive and do not require expensive and sophisticated detection systems. The most significant limitation of PCR-ARMS is that it can be used to detect only known mutations and polymor- phisms. As such, it is usually necessary to combine PCR- ARMS with other molecular diagnostic strategies (e.g. sequencing) to provide comprehensive mutation detec- tion. In its simplest formats, such as those described in this chapter, PCR-ARMS may be impractical for applications involving large numbers of mutations or high through- put. For such applications, consider using ARMS assays with allele detection strategies that are more amenable to automation. One such approach is to use ARMS primers labeled with fluorescent dyes. 2.3 PCR-ASO The analysis of point mutations in DNA using hybridiza- tion with ASO probes is based on the principle that even single nucleotide mismatches between a probe and its tar- get can destabilize the hybrid. ASO probes can be designed to be complementary and specific for the various alleles, thus providing a simple methodology to detect any known mutation or SNP. The use of ASO probes actually predates PCR, and was a commonly used approach to analyze cloned DNA. Radioactively labeled ASO probes have even been used to diagnose genetic disease using non-amplified genomic DNA that has been immobilized on a membrane after restriction endonuclease digestion and electrophoretic separation ( Conner et al. , 1983 ; Orkin et al. , 1983 ; Pirastu et al. , 1983 ). With the advent of PCR amplification ( Saiki et al. , 1985, 1988a, b ), PCR-ASO became one the first approaches used to analyze known point mutations within amplified DNA fragments ( Saiki et al. , 1986 ). 2.3.1 Basic Principles The design of ASO probes is largely dependent on the sequence of the region being targeted for analysis. ASO probes are generally short oligonucleotides (15- to 17- mers) with 30 – 50% G  C content, designed with the dis- criminating nucleotide located near the middle of the probe. Longer probes can be used to compensate for regions that have low G  C content. G:T and G:A mismatches are slightly destabilizing, whereas the effect is significantly FIGURE 2.3 Multiplex PCR-ARMS detection of 11 DHCR7 gene muta- tions. A. Schematic representation of the DHCR7 gene (introns not drawn to scale) showing the seven coding exons and PCR-ARMS strategies for detecting 11 point mutations associated with Smith – Lemli – Opitz syndrome. The mutations are detected in two multiplex PCR-ARMS assays (multiplex II mutations indicated in brackets). B. Analysis of PCR- ARMS multiplex products. Each multiplex includes an internal control amplicon from the HFE gene. Multiplex I: lane 1: normal control; lane 2: p.E448K carrier; lane 3: p.R404C; lane 4: p.W151X; lane 5: p.R352W; lane 6: p.T93M; lane 7: IVS 8-1 G  C. Multiplex II: lane 1: normal control; lane 2: p.R443C carrier; lane 3: p.T289I; lane 4: p.C380R; lane 5: p.V326L; lane 6: p.L109P. Chapter | 2 Allele-Specific Mutation Detection 21 and template DNA prevents extension of DNA synthesis, whereas in the competitive oligopriming system mismatch- ing prevents primer annealing (Table 2.1). 2.4.1 Basic Principles Differential primer annealing in competitive oligopriming is achieved by the use of three primers of DNA synthesis instead of two, in one polymerase chain reaction. A pair of competitive oligonucleotides is used as forward primers and the third primer is serving as a common reverse one, for the respective PCR. The mismatching is formed within the forward primer, usually in the middle, but in some cases is formed at the 3  end of the forward primer, as is the case of ARMS but in the competitive oligopriming setting, that is by using two competitive forward primers. The forward primers that detect DNA sequence altera- tions in competitive oligopriming are a pair of synthetic short DNA sequences (competitive oligoprimers or COP primers), carrying the mutant or the normal configuration at the mutation site in the middle of their sequence and capa- ble of discriminating between mutated and normal tem- plate DNA for annealing. Thus, competitive oligopriming is a system of allele-specific amplification, through differ- ential primer annealing. In competitive oligopriming allele- specific amplification occurs for both alleles in the same reaction, which is the important feature of the method. Once the competitive amplification of DNA has been completed, identification of the mutant versus the normal amplified allele among the products of a competitive PCR 2.3.3 Advantages and Limitations The PCR-ASO method, and particularly the reverse ASO format, provides a simple approach for simultaneous geno- typing of large numbers of mutations and polymorphisms. The method can be applied to any known sequence vari- ation, is non-radioactive, and does not require specialized instrumentation to detect the alleles. A potential drawback to the PCR-ASO strategy is the amount of developmental work needed to identify a panel of oligonucleotide probes that are allele specific under the same hybridization and wash stringency conditions. For small laboratories with limited resources, this initial investment may preclude the development of in-house PCR-ASO assays. For the same reasons, it is unlikely that commercial PCR-ASO will become available for rare diseases having limited market potential. 2.4 THE COMPETITIVE OLIGOPRIMING ASSAY Competitive oligonucleotide priming (COP) of DNA syn- thesis has been described for the first time by Gibbs and coworkers in 1989. As previously mentioned, COP is a strategy for the detection of known sequence varia- tions, based on allele-specific amplification, using ASO. However, there are two fundamental differences between the usual methods involving allele-specific amplification ( Nollau and Wagener, 1997 ) and competitive oligonucle- otide priming: in the former, mismatching between primer TABLE 2.1 Genetic loci for which the competitive oligopriming approach for mutation detection was applied. The position of the mismatch within COP primers, and the potential of multiplexing and/or high-throughput screening are recorded. HPRT : hypoxanthine phosphoribosyltransferase; BCHE : butyrylcholinesterase; ALDH2 : aldehyde dehydrogenase; PON : paraoxonase/arylesterase. Gene Mismatch position Multiplexing High throughput References HPRT Middle No No Gibbs et al. (1989) HBA2/HBA1, HBB Middle Yes No Chehab and Kan (1989) HBB Middle Yes No Athanassiadou et al. (1995) PON – Apolipo- protein B 3  end Yes Yes Germer and Higuchi (1999) ALDH2 3  end No No Koch et al. (2000) 3  end No Yes McClay et al. (2002) MCAD Factor V 3  end No No Giffard et al. (2001) Various SNPs 3  end No Yes Myakishev et al. (2001) BCHE Middle No No Yen et al. (2003) Molecular Diagnostics22 is rendered crucial. To this end a number of approaches have been applied, involving differential labeling of the competitive primers prior to their use in competitive PCR. Thus , the competitive oligopriming assay (COP assay) is carried out in two consecutive stages: the first stage involves the DNA amplification by competitive oligoprim- ing; the second stage involves the detection of the genetic identity – mutant versus normal – of the amplified material. 2.4.2 Genotyping with COP Assay 2.4.2.1 DNA Amplification In Vitro by COP In COP PCR, both allele-specific oligonucleotides – the mutated and the normal one – are used in the same reac- tion and through the competition that arises between them for annealing on the target DNA sequence, the binding of the perfectly matched primer to the template is strongly favored relative to the primer differing by a single base. Only one reverse primer is used in a competitive oligo- priming PCR, to serve synthesis with both allele-specific primers, which therefore must derive from the same – opposite of that of the reverse primer – strand of DNA sequence. One nevertheless can formulate a competitive PCR, in which the competitive oligoprimers are of opposite direction, but this necessitates the use of two reverse primers, too, and most probably elaborate optimization procedures. In COP PCR all three primers, namely the two forward primers for the mutation site, one (m) and the other (wt) and the common reverse primer, are used in one reaction ( Fig. 2.6 ) with a given template DNA. In this setting, competitive primers (m) and (wt) anneal only or mainly with the mutated and normal DNA, respec- tively, promoting correct priming and not vice versa. Results fall into three categories, depending on the geno- type of the template DNA: ● If the amplified DNA contains only (or mainly) the normal primer (wt), the template DNA used is homozygous wild-type for this site. ● If the amplified DNA contains only (or mainly) the mutated primer (m), the template DNA used is homozygous mutant for this site. ● If the amplified DNA contains both (m) and (wt) prim- ers in equal (or almost equal) amounts, the template DNA used is heterozygous for this site. The efficiency of COP PCR depends on a number of parameters but limited data exist on this issue: A) The primers ’ length : competition experiments between oligoprimers of different length have shown that 12-mers bear a greater potential for correct priming than 20-mers ( Gibbs et al ., 1989 ). However, the precise relationship between the length of competitive primers and their discriminatory potential depends on their overall sequence, as well as on the individual base mis- match ( McClay et al. , 2002 ). B) The annealing temperature : effective competition occurs at low stringency for shorter types of primers (12-mers) but annealing temperatures close to T m ( Yen et al. , 2003 ) or fairly stringent ( McClay et al. , 2002 ) to high stringent condition ( Athanassiadou et al. , 1995 ) gave good results with longer oligoprimers (16- to 20-mers). C) The primer concentration : an excess primer to template ratio, promoters correct priming in low as well as in high stringency reactions ( Athanassiadou et al. , 1995 ). D) The nature of the mismatch : successful competition has been shown for a number of mismatches between primer and template DNA, as documented in only one work ( Gibbs et al ., 1989 ). Finally, no data exist for the possible effect on COP PCR of the exact position of the mismatch within the COP oligoprimers. 2.4.2.2 Genotype Detection Systems The size of the PCR products in a COP PCR is the same in all three cases A, B, and C ( Fig. 2.6 ), that is irrespec- tive of the genotype of the PCR products and therefore a detection system of their genotype is necessary. This relies on the possibility for differential detection of the genetic identity (mutant vs normal) of the incorporated competi- tive primer and can be achieved by differential labeling of the two competitive primers. Various approaches have been employed so far: a color complementation assay was developed by Chehab and Kan (1989) , which allows dis- crimination between fluorescent oligonucleotide prim- ers. The addition of a 5  GC tail to one of the primers so that it can be distinguished by T m shift was employed by Germer and Higuchi (1999) , and allele-specific PCR with universal energy transfer labeled primers was developed by Myakishev and coworkers (2001) . Differential end labeling of the competitive oligoprimers with compounds that are recognized by different antibodies has also been applied ( Athanassiadou et al. , 1995 ). This approach was used for the differential identifica- tion of β -thalassemia mutation. In this system the detec- tion of the identity (normal vs mutant) of the COP primers incorporated in each case is carried out by means of differ- ential 5  labeling of the COP primers (mutant primer with dansylchloride and normal primer with FITC) that were subsequently recognized by specific antibodies on a solid support. The common reverse primer was biotinylated so as to facilitate the formation of a conjugate on the ampli- fied DNA. Three sets of results are shown (see Fig. 2.7c ), two sets for mutation HBB :c.93  21G  A (IVS I-110 G  A; I and II) and one for mutation HBB :c.92  6T  C (IVS I-6 T  C; III). The two sets of mutation HBB :c.93  21G  A Chapter | 2 Allele-Specific Mutation Detection 23 differ from each other only by 1 ° C in the annealing tem- perature; that is, case I is carried out at 1 ° C and case II at 2 ° C below high stringency. It is noticeable that a very low degree of mispriming persists for the n primer for mutation HBB :c.93  21G  A, like the practically negligible level of cross-hybridization of these primers when used in ASO hybridization, but this does not interfere with interpretation of results. The detection of β -thalassemia and other mutations is efficient and reliable with the COP assay applied and this leads to the conclusions that the COP assay, as exemplified here, is a robust and reliable method of mutation detection. FIGURE 2.6 The three possible outcomes of a competitive PCR using a pair of allele-specific competitive primers: dark dots (m): mutant, and dark squares (wt): normal; as well as a common reverse primer, with template DNA A. Wild type (dark squares only), B. Homozygous mutant (dark dots only), and C. Heterozygous (dark squares and dark dots). Competitive PCR products in each case show correct priming as a result of efficient competi- tion between the two primers for correct annealing on the template DNA. Dark and empty romvoids at the end of primers and PCR products represent differential labeling for the discrimination between normal and mutated amplified DNA. Molecular Diagnostics26 Cai , S.P. , Wall , J. , Kan , Y.W. , and Chehab , F.F. ( 1994 ) . Reverse dot blot probes for the screening of b-thalassemia mutations in Asians and American Blacks . Hum. Mutat. 3 , 59 – 63 . Chan , V. , Yam , I. , Chen , F.E. , and Chan , T.K. ( 1999 ) . A reverse dot-blot for rapid detection of non-deletion a thalassaemia . Br. J. Haematol. 104 , 513 – 515 . Chehab , F.F. ( 1993 ) . Molecular diagnostics: past, present, and future . Hum. Mutat. 2 , 331 – 337 . Chehab , F.F. , and Kan , Y.W. ( 1989 ) . Detection of specific DNA sequences by fluorescence amplification: a color complementation assay . Proc. Natl. Acad. Sci. USA, 86 , 9178 – 9182 . Chehab , F.F. , and Wall , J. ( 1992 ) . Detection of multiple cystic fibrosis mutations by reverse dot blot hybridization: a technology for carrier screening . Hum. Genet. 89 , 163 – 168 . Conner , B.J. , Reyes , A.A. , Morin , C. , Itakurs , K. , Teplitz , R.L. , and Wallace , R.B. ( 1983 ) . Detection of sickle cell S-globin allele by hybridization with synthetic oligonucleotides . Proc. Natl. Acad. Sci. USA, 80 , 278 – 282 . Eitan , Y. , and Kashi , Y. ( 2002 ) . Direct micro-haplotyping by multiple dou- ble PCR amplifications of specific alleles (MD-PASA) . Nucleic Acids Res. 30 , e62 . Foglieni , B. , Cremonesi , L. , Travi , M. , Ravani , A. , Giambona , A. , Rosatelli , M.C. , Perra , C. , Fortina , P. , and Ferrari , M. ( 2004 ) . Beta- thalassemia microelectronic chip: a fast and accurate method for mutation detection . Clin. Chem. 50 , 73 – 79 . Foglietta , E. , Bianco , I. , Maggio , A. , and Giambona , A. ( 2003 ) . Rapid detection of six common Mediterranean and three non-Mediterranean a-thalassemia point mutations by reverse dotblot analysis . Am. J. Hematol. 74 , 191 – 195 . Germer , S. , and Higuchi , R. ( 1999 ) . Single-tube genotyping without oli- gonucleotide probes . Genome Res. 9 , 72 – 78 . Gibbs , R.A. , Nguyen , P.-N. , and Caskey , T.C. ( 1989 ) . Detection of sin- gle DNA base differences by competitive oligonucleotide priming . Nucleic Acids Res. 17 , 2437 – 2448 . Giffard , P.M. , McMahon , J.A. , Gustafson , H.M. , Barnard , R.T. , and Voisey , J. ( 2001 ) . Comparison of competitively primed and conven- tional allele-specific nucleic acid amplification . Anal. Biochem. 292 , 207 – 215 . Gold , B. ( 2003 ) . Origin and utility of the reverse dot-blot . Expert Rev. Mol. Diagn. 3 , 143 – 152 . Huang , M.-M. , Arnheim , N. , and Goodman , M.F. ( 1992 ) . Extension of base mispairs by Taq DNA polymerase: implications for single nucle- otide discrimination in PCR . Nucleic Acids Res. 20 , 4567 – 4573 . Ikuta , S. , Tagaki , K. , Wallace , R.B. , and Itakura , K. ( 1987 ) . Dissociation kinetics of 19 base paired oligonucleotide-DNA duplexes contain- ing different single mismatched base pairs . Nucleic Acids Res. 15 , 797 – 811 . Koch , H.G. , McClay , J. , Loh , E.-W. , Higuchi , S. , Zhao , J.-H. , Sham , P. , Ball , D. , and Craig , I.W. ( 2000 ) . Allele association studies with SSR and SNP markers at known physical distances within a 1Mb region embracing the ALDH2 locus in Japanese, demonstrates linkage dis- equilibrium extending up to 400 kb . Hum. Mol. Genet. 9 , 2993 – 2999 . Kwok , S. , Kellogg , D.E. , McKinney , N. , Soasic , D. , Goda , L. , Levenson , C. , and Sninsky , J.J. ( 1990 ) . Effects of primer-template mismatches on the polymerase chain reaction: human immunodeficiency virus type 1 model studies . Nucleic Acids Res. 18 , 999 – 1005 . Makowski , G.S. , Nadeau , F.L. , and Hopfer , S.M. ( 2003 ) . Single tube mul- tiplex PCR detection of 27 cystic fibrosis mutations and 4 polymor- phisms using neonatal blood samples collected on Guthrie cards . Ann. Clin. Lab. Sci. 33 , 243 – 250 . McClay , J.L. , Sugden , K. , Koch , H.G. , Higuchi , S. , and Craig , I.W. ( 2002 ) . High-throughput single-nucleotide polymorphism genotyping by fluorescent competitive allele-specific polymerase chain reaction (SniPTag) . Anal. Biochem. 301 , 200 – 206 . Myakishev , M.V. , Khripin , Y. , Hu , S. , and Hamer , D.H. ( 2001 ) . High- throughput SNP genotyping by allele-specific PCR with universal energy-transfer-labeled primers . Genome Res. 11 , 163 – 169 . Newton , C.R. , Graham , A. , Heptinstall , L.E. , Powell , S.J. , Summers , C. , Kalsheker , N. , Smith , J.C. , and Markham , A.F. ( 1989 ) . Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS) . Nucleic Acids Res. 17 , 2503 – 2516 . Nichols , W.C. , Liepieks , J.J. , McKusick , V.A. , and Benson , M.D. ( 1989 ) . Direct sequencing of the gene for Maryland/German familial amyloidotic polyneuropathy type II and genotyping by allele-specific enzymatic amplification . Genomics 5 , 535 – 540 . Nollau , P. , and Wagener , C. ( 1997 ) . Methods for detection of point muta- tions: performance and quality assessment . Clin. Chem. 43 , 1114 – 1128 . Nowaczyk , M.J. , Nakamura , L.M. , Eng , B. , Porter , F.D. , and Waye , J.S. ( 2001 ) . Frequency and ethnic distribution of the common DHCR7 mutation in Smith – Lemli – Opitz syndrome . Am. J. Med. Genet. 102 , 383 – 386 . Okayama , H. , Curiel , D.T. , Brantly , M.L. , Holmes , M.D. , and Crystal , R.D. ( 1989 ) . Rapid nonradioactive detection of mutations in the human genome by allele-specific amplification . J. Lab. Clin. Med. 114 , 105 – 113 . Old , J.M. , Varawalla , N.Y. , and Weatherall , D.J. ( 1990 ) . Rapid detec- tion and prenatal diagnosis of b-thalassaemia: studies in Indian and Cypriot populations in the UK . Lancet 336 , 834 – 837 . Orkin , S.H. , Markham , A.F. , and Kazazian , H.H. , Jr. ( 1983 ) . Direct detec- tion of the common Mediterranean beta-thalassemia gene with syn- thetic DNA probes: an alternate approach for prenatal diagnosis . J. Clin. Invest. 71 , 775 – 779 . Patrinos , G.P. , Kollia , P. , and Papadakis , M.N. ( 2005 ) . Molecular diagno- sis of thalassemia and hemoglobinopathies: from RFLP analysis to microarrays . Hum. Mutat. 26 , 399 – 412 . Pirastu , M. , Kan , Y.W. , Cao , A. , Conner , B.J. , Teplitz , R.L. , and Wallace , R.B. ( 1983 ) . Prenatal diagnosis of b-thalassemia: detection of a single nucleotide mutation in DNA . N. Engl. J. Med. 309 , 284 – 287 . Polten , A. , Fluharty , A.L. , Fluharty , C.B. , Kappler , J. , von Figura , K. , and Gieselmann , V. ( 1991 ) . Molecular basis of different forms of meta- chromatic leukodystrophy . N. Engl. J. Med. 324 , 18 – 24 . Saiki , R.K. , Bugawan , T.L. , Horn , G.T. , Mullis , K.B. , and Erlich , H. A. ( 1986 ) . Analysis of enzymatically amplified b-globin and HLA- DQa DNA with allele-specific oligonucleotide probes . Nature 324 , 163 – 166 . Saiki , R.K. , Chang , C.-A. , Levenson , C.H. , Warren , T.C. , Boehm , C.D. , Kazazian , H.H. , Jr. , and Erlich , H.A. ( 1988 b ) . Diagnosis of sickle cell anemia and b-thalassemia with enzymatically amplified DNA and nonradioactive allele-specific oligonucleotide probes . N. Engl. J. Med. 319 , 537 – 541 . Saiki , R.K. , Gelfand , D.H. , Stoffel , S. , Scharf , S.J. , Higuchi , R. , Horn , G. T. , Mullis , K.B. , and Erlich , H.A. ( 1988 a ) . Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase . Science 239 , 487 – 491 . Saiki , R.K. , Scharf , S. , Faloona , F. , Mullis , K.B. , Horn , G.T. , Erlich , H.A. , and Arnheim , N. ( 1985 ) . Enzymatic amplification of b globin sequences and restriction site analysis for diagnosis of sickle cell anemia . Science 230 , 1350 – 1354 . Saiki , R.K. , Walsh , P.S. , Levenson , C.H. , and Erlich , H.A. ( 1989 ) . Genetic analysis of amplified DNA with immobilized sequence-specific oligo- nucleotide probes . Proc. Natl. Acad. Sci. USA, 86 , 6230 – 6234 . Chapter | 2 Allele-Specific Mutation Detection 27 Santacroce , R. , Ratti , A. , Caroli , F. , Foglieni , B. , Ferraris , A. , Cremonesi , L. , Margaglione , M. , Seri , M. , Ravazzolo , R. , Restagno , G. , Dallapiccola , B. , Rappaport , E. , Pollak , E.S. , Surrey , S. , Ferrari , M. , and Fortina , P. ( 2002 ) . Analysis of clinically relevant single-nucle- otide polymorphisms by use of microelectronic array technology . Clin. Chem. 48 , 2124 – 2130 . Sarkar , G. , Cassady , J. , Bottema , C.D.K. , and Sommer , S.S. ( 1990 ) . Characterization of polymerase chain reaction amplification of spe- cific alleles . Anal. Biochem. 186 , 64 – 68 . Sommer , S.S. , Cassady , J.D. , Sobell , J.L. , and Bottema , C.D. ( 1989 ) . A novel method for detecting point mutations or polymorphisms and its application to population screening for carriers of phenylketonuria . Mayo Clin. Proc. 64 , 1361 – 1372 . Sommer , S.S. , Groszbach , A.R. , and Bottema , C.D. ( 1992 ) . PCR ampli- fication of specific alleles (PASA) is a general method for rapidly detecting known single-base changes . Biotechniques 12 , 82 – 87 . Waterfall , C.M. , and Cobb , B.D. ( 2001 ) . Single tube genotyping of sickle cell anaemia using PCR-based SNP analysis . Nucleic Acids Res. 29 , e119 . Waye , J.S. , Nakamura , L.M. , Eng , B. , Hunnisett , L. , Chitayat , D. , Costa , T. , and Nowaczyk , M.J. ( 2002 ) . Smith – Lemli – Opitz syndrome: carrier frequency and spectrum of DHCR7 mutations in Canada . J. Med. Genet. 39 , e31 . Wu , D.Y. , Ugozzoli , L. , Pal , B.K. , and Wallace , R.B. ( 1989 ) . Allele-spe- cific enzymatic amplification of beta-globin genomic DNA for diagno- sis of sickle cell anemia . Proc. Natl. Acad. Sci. USA, 86 , 2757 – 2760 . Ye , S. , Dhillon , S. , Ke , X. , Collins , A.R. , and Day , I.N.M. ( 2001 ) . An efficient procedure for genotyping single nucleotide polymorphisms . Nucleic Acids Res. 29 , e88 . Yen , T. , Hightingale , N. , Burns , J.C. , Sullivan , D.R. , and Stewart , P. M. ( 2003 ) . Butyrylcholinesterase (BCHE) genotyping for post-suc- cinylcholine apnea in an Australian population . Clin. Chem. 49 , 1297 – 1308 . Zhang , Y. , Coyne , M.Y. , Will , S.G. , Levenson , C.H. , and Kawasaki , E.S. ( 1991 ) . Single-base mutational analysis of cancer and genetic diseases using membrane-bound modified oligonucleotides . Nucleic Acids Res. 19 , 3929 – 3933 . This page intentionally left blank Chapter | 3 Enzymatic and Chemical Cleavage Methods to Identify Genetic Variation 31 adsorbed onto silica beads under high salt concentrations (3 M TEAC solution) before undergoing the chemical mod- ification steps ( Bui et al., 2003a ). The post-cleavage wash- ing steps also were eliminated by incubation of piperidine together with the loading dye buffer. In addition, various types of amine-bases were also reported to improve cleav- age reactions used in CCM methods ( Block, 1999 ). All relevant protocols are suitable for analyzing mismatches located on long stretches of DNA (up to 2.0 kb). Recently, the cleavage step with piperidine was omitted in CCM as the permanganate oxidation of mismatched thymine and cytosine could be followed up via UV-visible spectroscopy ( Bui et al., 2003b ). This technique was very simple and suitable for short DNA heteroduplexes (up to 300 bp). For the purpose of practical guidance, this chapter describes the solid phase, liquid phase, and spectroscopic protocols which are most commonly used in our laboratories and oth- ers ( Gogos et al., 1990 ; Rowley et al., 1995 ; Roberts et al., 1997 ; Lambrinakos et al., 1999 ; Tabone et al., 2006 ). The formation of heteroduplexes can be performed by mixing and reannealing equimolar amounts of wild-type and mutant DNA, thereby resulting in mismatched base pairs. In principle, the imperfect duplexes are different from their corresponding perfect duplexes in terms of their local con- formational changes, physical and chemical properties ( Bui et al., 2002 ). These changes are reflected at lower melting temperatures in the imperfect duplexes ( Patel et al., 1982 ), less stability as indicated by thermodynamic constants ( Patel et al., 1982 ) and extra-helical or ‘ ‘ flip-out ’ ’ phenomena of mis- matched bases ( Kao et al., 1993 ; Roberts and Cheng, 1998 ). However, such discrepancies induced by mismatches are so small that the mismatched sites can be recognized and cleaved only by enzymatic means and not by chemical reagents ( Kennard, 1988 ). For this reason, the mismatched sites require further modification by chemicals before the chemical cleavage reaction (piperidine) can take place. Two common chemicals, KMnO 4 and NH 2 OH, are the most effective for modifying thymine and cytosine mismatches, respectively, in this regard. The former reaction leads to the formation of a mixture of a thymine glycol and a ketone analog (some evi- dence indicates that KMnO 4 also reacts with cytosine but to a lesser extent; Bui and Cotton, 2002 ) and the latter gives rise to a modified cytosine containing a hydroxylamine moiety (see Fig. 3.1 ). Physical data established on the model for short mismatched oligonucleotides (38 bp) indicate that the differ- ences in melting temperatures and gel mobility of the chemi- cally modified heteroduplex samples are significant compared to the unmodified one ( Bui et al., 2003b ). The results suggest that the destabilized mismatched site favors the site-selective cleavage reaction of piperidine and the mismatch can be pin- pointed on a denaturing gel as cleaved bands. Mechanism of the modification reaction by KMnO 4 is fully elucidated at molecular levels. KMnO 4 reacts with thymine and cytosine to form the complexes, which gave strong absorption at 420 nm. The formation of color intermediate was confirmed on solid support and formed a basis for a simple spectroscopic tech- nique to follow up the oxidation of mismatched DNA ( Bui et al., 2004 ). Proof of concept was carried out on synthetic 39 bp DNA fragments containing single base mismatches and the oxidation results with KMnO 4 clearly showed the color change, which could be detected by measuring the absorb- ance at 420 nm after 30 min reaction (end-point analysis) or by continuously scanning the reaction mixture over a period of time (scanning analysis). The mutation detection via spec- troscopy has been successfully applied in longer mismatched DNA (up to 300 bp) using UV/Vis microplate reader ( Tabone et al., 2006 ). The assay was validated as 100% effective in detection of single point mutation using a blind manner for all mismatched DNA samples, derived from the mouse β -globin gene promoter region, containing single point mutations as HN N O O O R N R N R N R O N R HN O OH OH KMnO4 HN O OH O N N O NH2 NH2-OH O HN OH Mismatched Thymine Mismatched Cytosine R = DNA backbone FIGURE 3.1 Chemical reactions involved in the CCM method. KMnO 4 selectively reacts with mismatched thymine to afford a mixture of thymine gly- col and a ketone analog, and hydroxylamine reacts with mismatched cytosine to afford a single monosubstituted product under the described conditions. Molecular Diagnostics32 well as patients who were previously screened for mutations by other methods (sequencing, SSCP or DHPLC analysis). In general, CCM and new versions (solid phase or spectro- scopic technique) are considered as the method of choice as it can detect all key types of mismatch (T/G, T/C, C/C, A/C, and T/T), which represent all eight possible mispairs that can be generated from the heteroduplex formation (see Fig. 3.2 ). The other mismatches (A/G, G/G, and A/A) can be detected via the complementary heteroduplexes (i.e. A/A will be detected by T/T). It is also emphasized that some neighboring matched bases also respond to the reactions due to instability of the whole region near the mismatched site. In addition, when both mutant and wild-type DNA are labeled, the chance of detecting mutations will be doubled (see Fig. 3.2 ). 3.3.1 Liquid Phase Protocol The standard liquid phase CCM protocol consists of six steps: chemical modification with KMnO 4 and hydroxy- lamine, termination, separation, washing, cleavage, and gel- electrophoresis (see Table 3.2 ). The wild-type and mutant DNA samples are amplified (by PCR) using the fluorescence- labeled primers (6-FAM and HEX at 5  A and 3  A ends, T T C TWT G C C TM C G G AM A A G AWT Heteroduplex 1 Heteroduplex 2 T T C T C G G A G C C TA A G A Wild-type Mutant  Melt & reanneal * # * * * # # # FIGURE 3.2 Formation of heteroduplexes from the wild-type (WT) and mutant (M) homoduplexes. * indicates 5  A-FAM and # indicates 3  A-HEX fluorescence-labeled DNA strands. TABLE 3.2 The liquid phase protocol. Steps Procedure a KMnO 4 reaction 0.2 mL of 100 mM KMnO 4  19.8 ml of 3 M TEAC. Incubate at 25 ° C for 10 min. Hydroxylamine reaction 20 mL of 4.2 M hydroxylamine solution (pH  6.0 with triethylamine). Incubate at 37 ° C for 40 min. Termination Add 200 ml of STOP buffer (25 mg/ml tRNA, 0.1 mM EDTA, 0.3 M sodium acetate, pH 5.2). Separation Add 750 ml of ice-cold 100% ethanol (at  20 ° C for at least 30 min). Centrifuge for 20 min at 14,000 rpm to collect the precipitates. Washing Rewash the pellet with 200 ml of 70% ethanol. Centrifuge it again for 10 min (14,000 rpm). Air-dry the pellet for 10 min. Cleavage Add 10 ml of cleavage loading dye solution (20 ml piperidine, 64 ml formamide and 16 ml dye (50 mg blue dextran in 1 ml distilled water)) to the DNA pellets and incubate at 90 ° C for 30 min. Gel separation b Load samples onto a denaturing gel and analyze on an ABI 377 DNA sequencer (2 ml of sample is needed for each well). a 6 ml of homoduplex and heteroduplex DNA (0.6 mg DNA) in TE buffer (10 mM Tris-HCl, 1 mM EDTA, ethylene-diamine-tetra-acetic acid, pH 8.0) is used for each of the reactions. b The denaturing polyacrylamide gel 4.25% (acrylamide: bis- acrylamide, 19:1), 6 M urea gel in the TBE buffer (16.2 g Tris-base, 8.1 g boric acid and 1.12 g EDTA in 1,500 ml distilled water, pH  8.0). Chapter | 3 Enzymatic and Chemical Cleavage Methods to Identify Genetic Variation 33 TABLE 3.3 The solid phase protocol. Steps Procedure Loading DNA onto solid supports Mix 3 ml of Ultra-bind bead suspension with 1 ml of DNA samples (0.1 to 0.2 mg of homoduplex or heteroduplex DNA) on shaker for 1 hour at 25 ° C. Centrifuge at 14,000 rpm, the pellets are collected. Wash the beads by resuspending in the Ultra-wash solutions (2  500 mL). Centrifuge the mixture, discard the supernatant, and air-dry the beads at 25 ° C for 15 min. KMnO 4 reaction Mix the beads with 0.3 ml of 100 mM KMnO 4 in 29.7 ml of 3 M TEAC solution. Allow to stand at 25 ° C for 5 min. Hydroxylamine reaction Mix the beads with 15 ml of 4.2 M hydroxylamine solution in 15 ml of 3 M TEAC solution. Allow to stand at 37 ° C for 40 min. Washing The beads are separated by centrifugation and washed twice with the Ultra-wash solution (200 mL per wash) and the pellets are air-dried at 25 ° C for 15 min. Cleavage Add 10 mL of the cleavage dye solution to each reaction tube and vortex well. Incubate the tubes at 90 ° C for 30 min. Mix tubes by flicking occasionally. Cool the tubes on ice and the supernatant is separated by centrifugation. Gel separation Load samples onto a denaturing gel and analyze on an ABI 377 DNA sequencer (2 ml of sample is needed for each well). respectively). Subsequently, the amplified DNA samples are purified, using either a commercially available purification kit (Stratagene ™ PCR Purification Kit, CA, USA) or agarose gel electrophoresis. The resulting wild-type and mutant DNA samples are mixed in equal amounts to form the heterodu- plexes prior to the assay. In the first step of the described pro- tocol, the reactions of heteroduplex DNA with KMnO 4 and hydroxylamine are usually carried out in separate tubes. It is also noted that both reactions can be optimized and carried out in a single tube protocol (but this will not be discussed in this chapter). When the reactions are completed, the DNA samples are separated by ethanol precipitation and washed carefully before the next cleavage step. To simplify the protocol, the gel loading dye (blue dextran) is added to the cleavage solution and the reaction mixtures can be directly loaded onto a denaturing gel immediately after the cleavage step. The DNA fragments are analyzed by an ABI 377-DNA sequencer without further purification (only 2 ml of sample is needed for loading). Two types of size standards (Tamra 500 and Tamra 2500) are added to the gel. Therefore, analysis with the ABI sequencer allows identification of the positions of the mismatches without the use of sequencing. 3.3.2 Solid Phase Protocol In order to bypass the separation and washing steps, the solid phase protocols have been developed by immobilizing DNA on silica solid supports (see Table 3.3 ). Attachment of DNA on silica in a high salt solution has been established and well practiced as an effective purification technique ( Bui et al., 2003a ). In the described protocol, the commer- cially available silica beads are used (MO BIO Laboratories Inc. CA, USA) to bind to the DNA, and the DNA-attached beads are then sequentially treated with chemicals, wash- ing solutions, and piperidine for cleavage. In the last cleav- age step, the DNA fragments are cleaved by piperidine and released simultaneously from the solid supports. The resulting supernatant is isolated and loaded directly onto a denaturing polyacrylamide gel. Mismatch detection is based on the comparison of the homoduplex and heteroduplex traces. A mutation is identi- fied by cleavage peaks present in the trace of heteroduplex sample but not in the control homoduplex sample. In the authors ’ laboratories, both liquid phase and solid phase protocols are routinely used for mismatch detection, and some typical examples are described next. The liquid and solid phase protocols were successfully carried out on 547 bp DNA fragments derived from the cloned mouse β -globin gene promoter DNA to detect T/C and T/G mismatches, respectively (see Figs 3.3 and 3.4 ). Detection of single base insertion and deletion (C base) was carried out with DNA fragments (893 bp in Fig. 3.5 and 660 bp in Fig. 3.6 ) derived from human mitochondrial DNA by using the liquid phase protocol with hydroxylamine reaction. 3.3.3 Spectroscopic Protocol Spectroscopic analysis is based on the increased oxida- tion level of the reaction of mismatched thymine and cyto- sine with potassium permanganate. In a typical protocol, Molecular Diagnostics36 500 bp DNA sample containing 18 consecutive A/T matched pairs (see Fig. 3.8 ). KMnO 4 susceptibility to AT- rich sequences was also suggested due to the local destack- ing nature (curvature) of the repeated A-track region ( De Santis et al., 1990 ). Finally , to improve the quality of the result, PCR products of mutant and wild-type DNA usually are purified prior to any subsequent treatment, and the KMnO 4 solution should be freshly made before use because an aging solu- tion turns brown – yellow with the precipitation of MnO2 after a few days. The new CCM version (spectroscopic technique) now becomes an even simpler and inexpensive assay for detection of mutations and polymorphisms as it does not require expensive and toxic chemicals as well as separation steps. 3.5 ENZYMATIC CLEAVAGE OF MISMATCH METHODS 3.5.1 The mutHLS System of Mismatch Detection The post-replicative mismatch repair enzyme systems were explored as potential tools for mismatch detection ( Lu and Hsu, 1992 ; Lishanski et al., 1994 ; Wagner et al., 1995 ). Because the MutHLS system is tightly coupled to the DNA replication machinery in vivo , it is not meant to screen the genome as a whole for the presence of mismatches. The crystal structure further illuminated that MutS bends the DNA only slightly, allowing it to recognize almost all of the mismatches, but not with very high specificity or affinity TABLE 3.4 The spectroscopic protocol. DNA heteroduplexes A420 nm (mismatched) A420 nm (control)a % Increase in oxidation level Mismatch T-C at the 19th base from 5  end 0.358 0.300 16% Mismatch T-T at the 19th base from 5  end 0.530 0.230 57% Mismatch T-G at the 19th base from 5  end 0.326 0.230 30% a Homoduplex (control) 5  -GGAAGAAGGCATACGGGTGAACTAGGGCAGCGGACAAT-3  3  - CCTTCTTCCGTATGCCCACTTGATCCCGTCGCCTGTTA-5  . FIGURE 3.7 Detection of C/C mismatch by the solid phase protocol. The heteroduplex DNA trace displays two strong cleavage peaks of the 3-HEX and 5  A-FAM sequences induced by hydroxylamine/piperidine reactions. The control trace is not shown. FIGURE 3.8 Multiple cleavage reactions at the AT-rich sequence by KMnO 4 /piperidine. The homoduplex DNA trace displays a plateau peak due to cleavage reactions at the AT-rich sequence. Chapter | 3 Enzymatic and Chemical Cleavage Methods to Identify Genetic Variation 37 ( Natrajan et al., 2003 ). As a result, mutation screening based on MutS protein has thus far been unsatisfactory. 3.5.2 DNA N-glycosylase Approaches of Mismatch Detection The DNA N-glycosylases have evolved nucleotide-binding pockets with very tight fit and nucleotide specificity, including the ability to insert a peptide loop into the base that has been displaced. Unfortunately, nature needs only two mismatches to be recognized by DNA glycosylases. The thymine DNA N-glycosylase recognizes the T/G mismatch that results from the deamination of 5-methyl- cytosine ( Hardeland et al., 2001 ), and the MutY and its homologs recognize the A/G mismatch that occurs from the misincorporation of 8-oxo guanine across from the A residue ( Sanchez et al., 2003 ). Thus, methods of mutation detection that use DNA glycosylases do not utilize all the mismatch heteroduplexes that are present during the PCR amplification of two alleles. Moreover, the DNA N-glyco- sylases do not detect insertion/deletion mutations, or muta- tions that do not form T/G and A/G mismatches. Because a DNA N-glycosylase only creates an apurinic site but will not break the DNA chain, the detection of DNA trun- cation at the mismatch site requires either the addition of an apurinic endonuclease or a base treatment step such as piperidine at elevated temperatures, and DNA cleanup steps. Double-stranded DNA breaks are not produced, thus requiring the use of denaturing conditions for the shortened single-stranded products to be analyzed. Successful appli- cation of the DNA glycosylase methods have been docu- mented in numerous publications ( Zhang et al., 2002 ). 3.5.3 The Resolvase Approach of Mismatch Detection Another enzymatic approach involves the use of DNA resolvases that recognize the DNA distortion created by mismatches mimicking the DNA recombination intermedi- ate structures. Successful applications of this approach use the T4 endonuclease VII system ( Mashal et al., 1995 ; Youil et al., 1995, 1996 ) and the related T7 endonuclease I sys- tem ( Babon et al., 2003 ). These enzymes cut the mismatch duplexes within a few nucleotides of the mismatch sites, and usually lead to double-stranded breaks in the heterodu- plex. The resolvases also lead to non-specific DNA cutting at some unknown DNA sequences and imperfect products of PCR reactions ( Norberg et al., 2001 ). 3.5.4 The Endonuclease V Plus Ligation Method Although endonuclease V itself is not very mismatch specific, its incision within one or two nucleotides of a mismatch base allows a DNA ligase to distinguish between the mismatch product nicks that cannot be ligated and the nicks made at non-mismatch sites that can be repaired by ligation ( Huang et al., 2002 ). Some sequences may con- tain mismatches that are not detectable by endo V or liga- table due to mismatch slippage. In common with other approaches, there is non-specific nicking at AT-rich regions, and the ligation repair reaction is lengthy. 3.5.5 The Plant Mismatch Endonuclease Method The enzymatic mutation detection approach that currently shows much potential for exploitation in a number of appli- cations is the plant mismatch endonucleases exemplified by the CEL I endonuclease of celery ( Oleykowski et al., 1998, 1999 ; Yang et al., 2000 ; Kulinski et al., 2000 ). Recently, the CEL I method of mutation detection was successfully applied to the whole mitochondria genome ( Bannwarth et al., 2006, 2008 ), to the screening of epidermal growth factor receptor mutations ( Janne et al., 2006 ), to 25 human mutations (Tsuji and Niida, 2007), for variants in ATM , TGFB1 , XRCC1 , XRCC3 , SOD2 , and hHR21 ( Ho et al., 2006 ), for ATRX gene mutation ( Wada et al., 2006 ), for TP53 ( Poeta et al., 2007 ), for Sult1a1 ( Greber et al., 2005 ), and for TILLING in rice ( Till et al., 2007 ) and Drosophila ( Winkler et al., 2005 ). These plant nucleases apparently belong to a subgroup in the S1 nuclease family and are induced during plant senescence and remodeling. Mutation detection is extremely simple, applicable on various frag- ment analysis platforms, whereas the detection of the prod- ucts of DNA truncation at the mismatch site in either the single-stranded form or the double-stranded forms gives the potential of multiple detection formats. Moreover, the high precision of the CEL I nuclease to cut at the 3  phosphodi- ester bond immediately next to the mismatched base allows DNA ligation repair to be used more effectively if desired. The latter was used in the early experiments during the devel- opment of the CEL I platform, but is not necessary for most CEL I applications. The high mismatch specificity of CEL I nuclease is believed to come from the enzyme binding to both bases of a base substitution mismatch at the same time. Current CEL I mutation detection assay is exemplified in Fig. 3.9 , in which a DNA heteroduplex contains a mis- match. The ability of CEL I to form a single-stranded DNA nick in short incubations but convert to double-strand DNA truncation mode under conditions of longer incubations or enzyme excess has allowed two powerful assay approaches to be developed. 3.5.5.1 Single-Stranded DNA Truncation Assay This assay mode is widely used in fragment analysis plat- forms like the ABI-377 slab gel system, the LiCor infrared Molecular Diagnostics38 slab gel system, the ABI-3100/3730 capillary DNA sequencers, and the Beckman CEQ8000 infrared DNA sequencer. In this assay, the PCR primers for a given target region, often under 600 bp long, are labeled with one color for the forward primer and a second color for the reverse primer. The color combination used for the 377 system is 6-FAM/TET, for the ABI 3100/3730 is 6-FAM/HEX, and for the Beckman CEQ 8000 is Cy5/Cy 5.5 or D3/D4. For the latter, the Cy5/Cy5.5 combination is easier in DNA synthesis and purification. Purification of the infrared dye primers is not necessary because the presence of reporterless primers enhance the PCR efficiency without diminishing the mutation detection sensitivity. As illustrated in Fig. 3.9 , the two strands will be differentially labeled after PCR and heteroduplexes will be formed, either during PCR or thereafter by denaturation and renaturation. The heterodu- plex is treated with CEL I, without further purification, for about 5 – 30 min. The cut DNA can be loaded onto a DNA sequencer/fragment analyzing system without further puri- fication. A purification step enhances the performance of some fragment analysis platforms like the CEQ 8000 and the LiCor sequencer. The CEL I truncation bands of two colors are measured on the fragment analysis system. The sum of the lengths of the bands of the two colors correlated to the same SNP is equal to the full-length PCR primer plus one nucleotide, or more if the insertion involves more than one base. CEL I cuts an insertion at the phosphodi- ester bond at the 3  end of the loop, and then shortens the single-stranded region slowly thereafter. This assay is sim- ple, sensitive, and easily automated for high throughput. The two color cuts each originate from a different DNA molecule in the case of the single-strand cut assay, and thus their presence represents independent confirmation of the presence of the mismatch. This assay is used routinely in BRCA1 and BRCA2 genetic screening at the Fox Chase Cancer Center, and in the TILLING reverse genetics proce- dure ( Colbert et al., 2001 ) which offers another method of targeted gene knockout in plants, zebra fish, mice, ES cells, and other organisms. An important point in the single-strand mismatch nick- ing assay is that the nicked DNA may be a minor population in the case of a weak mismatch substrate like the base sub- stitutions T/T and G/T mismatches. Although the insertion/ deletions produce restriction-enzyme-like strong signals with CEL I single-strand nicking assay, many base sub- stitutions require rescaling of the fluorescence intensity axis (see Figs 3.10 and 3.11 ). Experiments in Figs 3.11 and 3.12 use a 500 bp PCR product from the exon 11.4 of the BRCA1 gene ( Oleykowski et al., 1998 ). This human genomic fragment, containing two TZC base substitutions and one GZA base substitution, is a very demanding stand- ard routinely used in the authors ’ laboratory for comparison of the mutation detection performance of different assay systems and conditions. The CEL I mismatch-specific sig- nals may be small when the PCR product and the primer peaks are expressed in full scale (see Fig. 3.11 , insert), but they are still many times higher in signal than the back- ground peaks. Figure 3.11 illustrates that even in the case of three mismatches being present in the same PCR frag- ment, they are decisively identified in the CEL I mismatch detection assay on the CEQ 8000. The same performance was previously reported for the ABI 377 platform ( Kulinski et al., 2000 ), and also routinely obtained for the ABI 3100 capillary sequencing system (see Fig. 3.12 ). A PCR product from two alleles form heteroduplexes of two combinations for any given base substitution (e.g. a CZT transition produces both C/A and T/G mismatches and four recognition sites for CEL I). Because CEL I mismatch nuclease can recognize all mismatches and can cut at either strand or both strands of a mismatch, all mismatches have multiple chances to be detected with this system. When used carefully, this system does not produce false positives and false negatives. Table 3.5 shows the assay conditions used for the CEQ 8000 system (Yeung laboratory) and the conditions rou- tinely used for BRCA1/BRCA2 mutation detection on the ABI 3100 system (Godwin laboratory), respectively. The CEQ 8000 system apparently produces slightly sharper peaks than the ABI 3100, but is less tolerant to salt and primer contaminations. Therefore, an ethanol precipitation step is needed prior to loading of the CEL I reaction prod- ucts onto the CEQ 8000. On the contrary, the ABI 3100 5’ 5' PCR product heteroduplex  CEL I 0.1−0.025 unit Fragment analysis native or denaturing conditions Optional cleanup5-30 min 45 C Y Z FIGURE 3.9 Schematic of the CEL I mismatch nuclease mutation detection procedure. The top panel illustrates that in a DNA heteroduplex containing a mismatch Y/Z, CEL I makes either one or two incisions in the same DNA molecule denoted by the two arrows. The bottom panel illustrates the sim- plicity of the CEL I mutation detection method. Adenaturing fragment analysis method may use 5  termini radioactive labeling or fluorescence two-color labeling followed by resolution of an automated DNA sequencer. The non-denaturing fragment analysis methods may use SYBR-green fluorescence staining and resolution on an agarose gel or PAGE or an Agilent BioAnalyzer lab-on-a-chip. TABLE 3.5 CEL I mismatch nuclease single-strand mismatch nicking protocols for capillary DNA sequencers. Capillary sequencer CEL protocols Yeung laboratory, Beckman CEQ 8000 Godwin laboratory, ABI 3100 PCR reaction 20 μ L 20 μ L Human genomic DNA 60 – 75 ng 75 – 150 ng Reaction buffer, 10  MgCl 2 , 2 mM final MgCl 2 DMSO 5% final 6.7% final dNTP 200 μ M 80 μ M Primers, each 1.5 pmol 4 pmol DNA polymerase 1U AmpliTaq Gold 1U AmpliTaq PCR cycles 94 ° C for 4 min 94 ° C for 4 min 97 ° C for 1 min 20 cycles of: Exons that are prone to deletion are checked with agarose gels 94 ° C for 5 sec 65 ° C for 1 min, touchdown at  0.5 ° C/cycle 72 ° C for 1 min 30 cycles of: 32 cycles of: 94 ° C for 10 sec 94 ° C for 5 sec 55 ° C for 20 sec 55 ° C for 1 min 72 ° C for 45 sec 72 ° C for 1 min 72 ° C for 4 min, then 4 ° C 72 ° C for 5 min, then 4 ° C Denaturation/renaturation step 94 ° C 1 min then cooled to 4 ° C over 30 min None CEL I reaction 20 μ L rxn 10 μ L rxn PCR reaction product 5 μ L 5 μ L Water 12 μ L 3.1 μ L 10  CEL I buffer 2 μ L 0.9 μ L CEL I enzyme 0.1 units 0.025 units Incubation 45 ° C for 7 min 45 ° C for 1 hr Stopping CEL I reaction 5 μ L of (0.1 mM EDTA, 1.2 M Na-acetate, 15 v/v glycogen, 1.7% v/v of NF co-precipitant) to the 20 μ L CEL I rxn Add 3 μ L 5 mM o-phenantholine or just add formamide tracking dye. No ethanol ppt or centrifugation 60 μ L of 95% ethanol, to a final volume of 85 μ L. Centrifuge for 15 min 4 ° C at 21,000  g Wash pellet 2x with 70% ethanol, dry in speedvac Preparation for capillary Resuspend pellet in 40 μ L of sample loading solution (SLS) 5 μ L of stopped enzyme rxn is added to 15 μ L of marker mix Sequencer loading Add 1.5 μ L of suspension to 28.5 mL of SLS. Add 10 μ L marker mix Load all 40 μ L to the CEQ8000 Load all 20 μ L to the ABI 3100 Sample well  0.25 μ L of the original PCR reaction Sample well approximately 1.92 μ L of the reaction original PCR Molecular Diagnostics42 native PAGE visualized with SYBR-green fluorescence staining. A second benefit of this native gel approach is that any single-stranded non-specific nicking by CEL I in the double-stranded DNA does not contribute to back- ground unless it leads to double-stranded truncation. Double-stranded truncation at a mismatch by CEL I can be obtained by using the same amount of CEL I as in the single-strand nicking assay but extending the incubation for 16 hours. The use of a higher concentration of CEL I for a shorter incubation produces much higher signal-to- noise ratio. The new CEL nuclease SURVEYOR ™ agar- ose gel kit supplied by Transgenomic Inc. has been further optimized for this assay and can detect base substitutions in long PCR products (i.e. 4 kb in length). For shorter frag- ments, a pool depth of one mutant in 40 normal alleles can be obtained. For biological DNA, the Giraff approach has demonstrated mismatch detection in heteroduplexes longer than 10 kb ( Sokurenko et al., 2001 ). The full exploitation of the agarose/acrylamide gel assay format is still in progress. For example, the use of the Agilent BioAnalyzer lab-on-a-chip system for this assay would produce automation and very sensitive detection of PCR products up to 12 kb, provides options for back- ground subtraction, and takes less than an hour. Because the proofreading DNA polymerase Optimase is used in the SURVEYOR ™ kit, the PCR products can be longer, while it allows trans-intronic PCR reactions to be performed for inbred strains. Besides leading to higher screening efficiency, longer PCR products also allow the position- ing of the query region near the center of the PCR product, thereby making this assay easy and efficient even when an ordinary inexpensive agarose gel apparatus is used. This mutation detection format is truly fast, convenient, and amenable to automation because no harsh chemicals are used, all enzyme steps can be done in the same tube, it takes little over an hour, and processing of the CEL nucle- ase incised DNA is unnecessary prior to loading onto the native condition fragment analysis systems. 3.5.5.3 Endonucleolytic Mutation Analysis by Internal Labeling (EMAIL) The 5  exo/endonuclease activity of the CEL family of nucleases is a major limitation on the ease of use of the CEL nuclease mutation detection method when 5  reporter labeling approaches are used. Recently, internally labeling by polymerase to incorporate a small amount of fluores- cently labeled dNTP during the PCR reaction was shown to lead to a DNA heteroduplex product in which the fluores- cence reporter is protected by the CEL nuclease and thus lead to higher assay sensitivity ( Cross et al., 2008 ). The mutation detection assay was conveniently performed on a 3730 DNA Analyzer (Applied Biosystems). 3.5.5.4 The s-RT-MELT Method Combining CEL Nuclease Enzymatic Selection with Real-Time DNA-Melting Curve Detection Most CEL nuclease mutation methods are gel-based. Recently, a method that does not involve the use of gel electrophoresis has been developed ( Li et al., 2007 ). This method, s-RT-MELT, scores for the appearance of CEL nuclease incision in heteroduplex DNA by the effect of the incision on the melting curve of the DNA duplex by using a real-time PCR instrument to perform a melting curve analysis. Besides the possibility of high throughput by using the microtiter plate formats, the assay employs ligation-mediated PCR reaction to amplify the CEL nucle- ase cut fragment, with PCR primer designs that enrich for the CEL nuclease-incised fragments. This cleaver assay for- mat is compatible with robotics and thus has the potential to greatly enhance the throughput of enzymatic mutation detection. 3.6 CONCLUSIONS There has been dramatic improvement in the methods for screening for unknown mutations. These improve- ments have already translated into exciting applications in numerous research centers. For both the chemical and the enzymatic methods of mutation detection, PCR artefacts FIGURE 3.13 CEL I mutation detection double-strand DNA truncation assay of SNPs in the BRCA1 gene. Each PCR product was incubated with 0.025 units of CEL I for 17 hours at 37 ° C and resolved on 8% native PAGE in 1X TBE buffer. DNA bands were visualized with SYBR-green fluorescence staining. Lanes 1, 100 base marker; 2, 1 kb marker; 3, Exon 2 control; 4, Exon 2 AG deletion; 5, Exon 20 control; 6, Exon 20 C inser- tion; 7, Exon 11.9 control; 8, Exon 11.9 4 bp deletion; 9, Exon 11.4 con- trol; 10, Exon 1 1.4 G A base substitution. Chapter | 3 Enzymatic and Chemical Cleavage Methods to Identify Genetic Variation 43 and DNA polymerization errors often limit the sensitivity these assays can deliver. One approach that potentially can remove these errors is the use of hairpin primers in PCR that effectively link each pair of semiconservative DNA replication strands as one duplex ( Kaur and Makrigiorgos, 2003 ). Any duplex that contains a PCR error can be removed with some mismatch-specific method to be deter- mined so that error-free PCR products can be collected and used in high-sensitivity mutation detection. It is expected that more improvements will be forthcoming so that effec- tive mutational screening will become a routine research and diagnostic tool for modern genetics. REFERENCES Babon , J.J. , McKenzie , M. , and Cotton , R.G. ( 2003 ) . The use of resolvases T4 endonuclease VII and T7 endonuclease I in mutation detection . Mol. Biotechnol. 23 , 73 – 81 . Bannwarth , S. , Procaccio , V. , and Paquis-Flucklinger , V. ( 2006 ) . Rapid identification of unknown heteroplasmic mutations across the entire human mitochondrial genome with mismatch-specific Surveyor Nuclease . Nat. Protoc. 1 , 2037 – 2047 . Bannwarth , S. , Procaccio , V. , Rouzier , C. , Fragaki , K. , Poole , J. , Chabrol , B. , Desnuelle , C. , Pouget , J. , Azulay , J.P. , Attarian , S. , Pellissier , J.F. , Gargus , J.J. , Abdenur , J.E. , Mozaffar , T. , Calvas , P. , Labauge , P. , Pages , M. , Wallace , D.C. , Lambert , J.C. , and Paquis-Flucklinger , V. ( 2008 ) . Rapid identification of mitochondrial DNA (mtDNA) mutations in neuromuscular disorders by using surveyor strategy . Mitochondrion 8 , 136 – 145 . Block, W. (1999). Improved detection of mutations in nucleic acids by chemical cleavage. Patent Appl. No. PCT/ US98/16385. Bui , C.T. , and Cotton , R.G.H. ( 2002 ) . Comparative study of permanga- nate oxidation reactions of nucleotide bases by spectroscopy . Bioorg. Chem. 30 , 133 – 137 . Bui , C.T. , Rees , K. , Lambrinakos , A. , Bedir , A. , and Cotton , R.G.H. ( 2002 ) . Site selective reactions of imperfectly matched DNA with small chemical molecules: applications in mutation detection . Bioorg. Chem. 30 , 216 – 232 . Bui , C.T. , Lambrinakos , A. , Babon , J.J. , and Cotton , R.G.H. ( 2003 a ) . Chemical cleavage reactions of DNA on solid support: application in mutation detection . BMC Chem. Biol. 3 , 1 . Bui , C.T. , Lambrinakos , A. , and Cotton , R.G.H. ( 2003 b ) . Spectroscopic study in permanganate oxidation reactions of oligonucleotides con- taining single base mismatches . Biopolymers 70 , 628 – 636 . Bui , C.T. , Sam , L.A. , and Cotton , R.G.H. ( 2004 ) . UV-visible spectral identification of the solution- phase and solid-phase permanganate oxidation reactions of thymine acetic acid . Bioorg. Med. Chem. Lett. 14 , 1313 – 1315 . Burdon , M.G. , and Lees , J.H. ( 1985 ) . Double-strand cleavage at a 2- base deletion mismatch in a DNA heteroduplex by Nuclease-S1 . Bioscience Reports 5 , 627 – 632 . Colbert , T.G. , Till , B. , Tompa , R. , Reynolds , S.H. , Steine , M. , Yeung , A.T. , McCallum , C.M. , Comai , L. , and Henikoff , S. ( 2001 ) . High-throughput screening for induced point mutations . Plant Physiol. 126 , 480 – 484 . Cotton , R.G.H. , Rodrigues , H.R. , and Campbell , R.D. ( 1988 ) . Reactivity of cytosine and thymine in single base-pair mismatches with hydroxy- lamine and osmium tetroxide and its application to the study of muta- tions . Proc. Natl. Acad. Sci. USA, 85 , 4397 – 4401 . Cotton , R.G.H. ( 1999 ) . Detection of mutations in DNA and RNA by chemical cleavage . In The Nucleic Acid Protocols Handbook Methods in Molecular Biology Series ( Rapley , R. , ed.) , pp. 685 – 693 . John Wiley & Sons, Inc . Cotton , R.G.H. , and Bray , P.J. ( 2001 ) . Using CCM and DHPLC to detect mutations in the glucocorticoid receptor in atherosclerosis: a compari- son . J. Biochem. Biophys. Methods, 47 , 91 – 100 . Cross , M.J. , Waters , D.L. , Lee , L.S. , and Henry , R.J. ( 2008 ) . Endonucleolytic mutation analysis by internal labeling (EMAIL) . Electrophoresis . in press . De Santis , P. , Palleschi , A. , Savino , M. , and Scipioni , A. ( 1990 ) . Validity of the nearest-neighbor approximation in the evaluation of the elec- trophoretic manifestations of DNA curvature . Biochemistry 29 , 9269 – 9273 . Gogos , J.A. , Karayiorgou , M. , Aburatani , H. , and Kafatos , F.C. ( 1990 ) . Detection of single base mismatches of thymine and cytosine residues by potassium permanganate and hydroxylamine in the presence of tetralkylammonium salts . Nucleic Acids Res. 18 , 6807 – 6812 . Greber , B. , Tandara , H. , Lehrach , H. , and Himmelbauer , H. ( 2005 ) . Comparison of PCR-based mutation detection methods and applica- tion for identification of mouse Sult1a1 mutant embryonic stem cell clones using pooled templates . Hum. Mutat. 25 , 483 – 490 . Hardeland , U. , Bentele , M. , Lettieri , T. , Steinacher , R. , Jiricny , J. , and Schar , P. ( 2001 ) . Thymine DNA glycosylase . Prog. Nucleic Acid Res. Mol. Biol. 68 , 235 – 253 . Haris , I.I. , Green , P.M. , Bentley , D.R. , and Giannelli , F. ( 1994 ) . Mutation detection by fluorescent chemical cleavage: application to hemophilia B . PCR Meth. Appl. 3 , 268 – 271 . Hartge , P. , Struewing , J.P. , Wacholder , S. , Brody , L.C. , and Tucher , M.A. ( 1999 ) . The prevalence of common BRCA1 and BRCA2 mutations among Ashkenazi Jews . Am. J. Hum. Genet. 64 , 963 – 970 . Ho , A.Y. , Atencio , D.P. , Peters , S. , Stock , R.G. , Formenti , S.C. , Cesaretti , J.A. , Green , S. , Haffty , B. , Drumea , K. , Leitzin , L. , Kuten , A. , Azria , D. , Ozsahin , M. , Overgaard , J. , Andreassen , C.N. , Trop , C.S. , Park , J. , and Rosenstein , B.S. ( 2006 ) . Genetic predictors of adverse radiotherapy effects: the Gene-PARE project . Int. J. Radiat. Oncol. Biol. Phys. 65 , 646 – 655 . Huang , J. , Kirk , B. , Favis , R. , Soussi , T. , Paty , P. , Cao , W. , and Barany , F. ( 2002 ) . An endonuclease/ligase based mutation scanning method especially suited for analysis of neoplastic tissue . Oncogene 21 , 1909 – 1921 . Janne , P.A. , Borras , A.M. , Kuang , Y. , Rogers , A.M. , Joshi , V.A. , Liyanage , H. , Lindeman , N. , Lee , J.C. , Halmos , B. , Maher , E.A. , Distel , R.J. , Meyerson , M. , and Johnson , B.E. ( 2006 ) . A rapid and sensitive enzy- matic method for epidermal growth factor receptor mutation screen- ing . Clin. Cancer Res. 12 , 751 – 758 . Kao , J.Y. , Goljer , I. , Phan , T.A. , and Bolton , P.H. ( 1993 ) . Characterization of the effects of a thymine glycol residue on the structure, dynam- ics and stability of duplex DNA by NMR . J. Biol. Chem. 268 , 17787 – 17793 . Kaur , M. , and Makrigiorgos , G.M. ( 2003 ) . Novel amplification of DNA in a hairpin structure: towards a radical elimination of PCR errors from amplified DNA . Nucleic Acids Res. 31 , e26 . Kennard , O. ( 1988 ) . Structural studies of base pair mismatches and their relevance to theories of mismatches formation and repair . In Structure and Expression, Vol. 2 in DNA and its Drugs Complexes ( Sarma , R. H. , and Sarma , M.H. , eds) , pp. 1 – 25 . Adenine Press . Kulinski , J. , Besack , D. , Oleykowski , C.A. , Godwin , A.K. , and Yeung , A.T. ( 2000 ) . The CEL I Enzymatic Mutation Detection Assay . Biotechniques 29 , 44 – 48 . Molecular Diagnostics46 SSCP analysis is extremely advantageous for fast muta- tion screening of known loci because it is easy to use and can preclude the use of radioactive substances for detection (see also sections 4.3 and 4.4). On the other hand, SSCP is not considered the method of choice for the analysis of unknown sequences, as there is no theoretical background established so far that could enable one to predict the exact electrophoretic mobility of a given DNA fragment accord- ing to its sequence (as is the case with denaturing gradi- ent gel electrophoresis (DGGE; see also Chapter 6) ). It is noteworthy that SSCP has already enjoyed an enormous success in mutation screening and it is still the method of choice in many molecular genetic laboratories. 4.3 FLUORESCENT SINGLE STRAND CONFORMATION POLYMORPHISM ANALYSIS Fluorescent SSCP (F-SSCP) is a non-radioactive high- resolution PCR-SSCP method, in which fluorescently labeled PCR products are electrophoresed and detected by an automated DNA sequencer ( Makino et al. , 1992 ). A pro- cedure for fluorescence labeling of the PCR product and detection of bands of double-stranded DNA in polyacry- lamide gel illuminated with UV-light has been described previously ( Chehab and Kan, 1989 ). However, the sensitiv- ity of this method was not high enough for SSCP analysis. Therefore, coupling SSCP analysis with an automated DNA sequencer enables highly sensitive fragment detection. Use of an automated DNA sequencer also permits strict con- trol of any desired temperature, a fundamental requirement of SSCP analysis to obtain reproducible results. This way, fragment separation patterns can be highly reproducible at any ambient condition. In certain cases, variations in the mobility patterns have been reported ( Makino et al. , 1992 ). A solution to this problem is normalization between lanes by including an internal standard fragment labeled with a different fluorescent dye in each lane ( Ellison et al. , 1993 ; Iwahana et al. , 1994 ). Co-electrophoresis of an internal size standard DNA labeled with a dye different from that of the sample DNA allows the relative fragment mobility of sam- ple DNAs to be reproducibly determined using mobility of the internal standard DNA as a reference. F -SSCP results can be also quantitatively interpreted. This is feasible since the bands are detected as peaks in the fluorogram and their heights are proportional to the inten- sity of the fluorescence in a wide dynamic range ( Fig. 4.3 ). The direct entry of data into a computer allows objective interpretation of the results, while data from multiple sam- ples can be processed for further analysis, using specialized analysis software with a reduced possibility of error. These features can be particularly important for pedigree analysis and linkage studies of polymorphic DNA markers detected by SSCP analysis. FIGURE 4.1 Schematic representation of the SSCP principle. Amplified DNA for both the wild-type and mutant alleles is subjected to denatura- tion and then to immediate cooling, where the denatured single-stranded alleles adopt a specific conformation. As the single-stranded wild-type and mutant alleles have different conformations, they can be distinguished when electrophoresed through a native polyacrylamide gel. FIGURE 4.2 Schematic drawing of a typical result from an SSCP analysis. After staining, the gel can be divided into two parts: the part with the bands corresponding to the single-stranded alleles (SS) and the other part with the double-stranded bands (DS), as complete denatura- tion is sometimes not feasible. In this example, lane 1 corresponds to the wild-type pattern for any given locus analyzed. If lane 2 corresponds to a homozygous case for mutation A, then lane 3 corresponds to a hetero- zygous case for the same mutation, and lanes 4 and 5 correspond to a homozygous case for mutation B and a compound heterozygous case for mutations A and B, respectively. Lanes 6 to 8 are characteristic examples of the electrophoretic pattern of small deletions or insertions, which can also be distinguished at the DS part of the gel (see also Fig. 4.5b). Lanes 6 and 7, heterozygous and homozygous cases for a small deletion, respec- tively. Lane 8, heterozygous case for a small insertion. Chapter | 4 Mutation Detection by Single Strand Conformation Polymorphism and Heteroduplex Analysis 47 The first generation F-SSCP was employing gel-based DNA sequencers. The Pharmacia ALF DNA sequencer was the instrument of choice as, at that time, it was the only commercially available fluorescence-based DNA sequencer equipped with an electrophoresis apparatus that allowed accurately controlled electrophoresis temperature. With the advent of capillary electrophoresis (see also next chapter), multicolor fluorescently labeled DNA fragments were PCR amplified in a single tube reaction and directly subjected to SSCP analysis using an automatically oper- ated capillary electrophoretic system (CE-SSCP; Inazuka et al. , 1997 ). In principle, any automated capillary or gel electrophoresis system can be used for F-SSCP, although the Applied Biosystems (Foster City, CA, USA) and Pharmacia (Uppsala, Sweden) ALF automated sequencers are more frequently employed. F -SSCP advantages include: 1. Non-radioactive labeling of the PCR products using different fluorescent dyes, allowing for highly sensitive and reproducible electrophoretic pattern detection and direct data storage and processing using specialized software; 2. Automated reloading of samples, allowing their electrophoresis in multiple temperatures which results in higher sensitivity; 3. Precise temperature control, resulting in higher sensi- tivity and greater reproducibility; 4. Fragment mobility normalization with the use of inter- nal lane control, allowing more accurate detection of mutations resulting in minor mobility alterations; 5. Lower running costs than conventional SSCP analy- sis, particularly if a large number of samples are to be analyzed in the long term with a throughput of several hundred samples per day, depending on the number of capillaries of the automated DNA sequencer; 6. Multiplexing, resulting in an increase in the analysis throughput. Multiplexing should include PCR products differing by at least 15 bp in size ( Ellison, 1996 ), and provided that the latter fragments are previously ana- lyzed individually in separate lanes to ensure that their electrophoretic patterns do not overlap. Minor disadvantages include the requirement of an expen- sive infrastructure, namely the automated DNA sequencer, while the size range for sensitive mutation detection has not been significantly extended. Therefore, similar to conven- tional SSCP analysis, PCR products only up to 500 bp can be efficiently analyzed using F-SSCP with a recommended size of 100 – 350 bp. 4.4 PARAMETERS INFLUENCING SINGLE STRAND CONFORMATION POLYMORPHISM ANALYSIS There are several parameters of SSCP analysis influencing the pattern of single strand conformation that will even- tually affect electrophoretic migration. These are DNA amplification (such as the size of PCR products), dena- turation, and the electrophoretic conditions (the length and duration of gel run, the ionic strength in the buffer being used, the temperature as well as the gel matrix composi- tion). Another important aspect of SSCP analysis is the visualization of the single-stranded DNA fragments, using a number of detection methods. All these aspects will be discussed in detail in the following paragraphs. 4.4.1 DNA Amplification Certain key aspects during amplification of the DNA frag- ment in question are of great importance in the performance of the SSCP analysis. Preferably, the size of the DNA frag- ment to be amplified should be between 150 and 350 bp. With certain exceptions, where mutation detection results have been reported for fragments of approximately 550 bp, this range is relatively safe and has reached a consensus between different laboratories ( Hayashi, 1991 ; Hayashi and Yandell, 1993 ). Typically, one should start to screen DNA fragments that will lie within the range of 100 to 350 bp, although this is often dependent on the sequence of the DNA fragment, the GC content, as well as the various elec- trophoretic conditions being used. However, if longer PCR fragments are to be screened, it is advisable to digest the fragment with restriction enzymes. The choice of restric- tion enzymes may affect considerably the resulted confor- mation and hence the performance of the SSCP assay. In general, the longer the fragments, the harder it is for any FIGURE 4.3 Principle of F-SSCP analysis. Wild-type DNA is repre- sented with the thick line and run in each lane as a control, along with the normal (Nl), heterozygous (Het) and homozygous (Hom) samples (thin line). Primers tagged with different fluorescent dyes were employed. Molecular Diagnostics48 given single nucleotide change to have an effect in the conformation of the fragment, although under certain cir- cumstances detection efficiency is not uniformly decreased with increasing DNA fragment length. It is noteworthy that sensitivity of SSCP can be greatly improved even for frag- ments as big as 800 bp, by running the electrophoresis in low pH buffer systems and at a fixed temperature ( Kukita et al. , 1997 ). After amplification, all PCR products should be screened on an agarose gel for the desired product length. If, despite considerable efforts, undesired side products are difficult to eliminate, a PCR product purification protocol should be applied. In addition, a negative and, where possible, a positive control of each PCR product should be included during each amplification step, since this will be the only indirect evidence that the screening assay is performed on the desired PCR products. PCR products can be stored on DNase-free tubes at 4 ° C. However, prolonged storage should be avoided and the subsequent steps of SSCP protocol should be performed as soon as possible. 4.4.2 Denaturation In SSCP analysis, it is important to achieve complete and as much irreversible denaturation of the DNA strands as possible. Incomplete denaturation, partial folding, and reannealing to double-stranded DNA will greatly reduce the amount of single-stranded DNA in the assay and will subsequently affect detection of the single-stranded mol- ecules. Usually, denaturation of the PCR products is car- ried out by incubation to high temperature, that is, 95 ° C for 5 to 7 min, and immediately after are chilled on ice for approximately 10 min. Alternatively, there are numerous denaturing agents, such as formamide, methyl-mercuric hydroxide, sodium hydroxide, and urea, which seem to per- form well ( Humphries et al. , 1997 ). Formamide is the most commonly used denaturing agent. In certain conditions, it is advisable to use 5 – 10% glycerol prior to loading the samples on the gel. This strategy was previously shown to produce sharper DNA bands, which greatly facilitate sub- sequent interpretation of results. Notably, an alternative method to increase the concentration of the single-stranded DNA molecules is asymmetric PCR. Following the first amplification step, an aliquot of the PCR product is used as template for nested PCR using only one of the primers employed in the previous amplification round ( L á zaro and Estivill, 1992 ). This approach overcomes the incomplete DNA denaturation and reannealing. 4.4.3 Electrophoretic Parameters Prior to reviewing the various electrophoretic parameters that may influence the SSCP analysis, it is worthwhile mentioning that currently there is neither adequate theory nor any physicochemical model available that could allow one to predict the three-dimensional structure of any given single-stranded DNA fragment, and as a result, its electro- phoretic mobility. Apart from the size of the DNA frag- ment and its GC content, the following parameters have been empirically found to affect the sensitivity of SSCP analysis: the gel matrix composition, the buffer composi- tion (ionic strength, the pH and buffer supplements, such as glycerol), the duration of gel run, the gel length, the DNA concentration, and the electrophoresis temperature. The effect of these factors on SSCP resolution and sensitivity is outlined next. 1. Gel matrix composition . For conventional SSCP analy- sis, the most common and widely accepted matrix is a cross-linked acrylamide polymer (8 – 12%). The small pore size of acrylamide-derived matrices makes it ideal for enhanced resolution and discrimination even at the nucleotide level. Higher resolution can be achieved upon addition of 10 – 15% of sucrose or glycerol. It has been previously shown that the mutation detection enhancement (MDE) gel (FMC Bioproducts) had a mutation detection rate of approximately 95% ( Ravnik- Glavac et al. , 1994 ). Although there is a considerable variability in the percentage of mutations detected with this commercially available gel matrix, it should be noted that in a considerable and rather growing number of studies for which SSCP has been implemented, MDE gels were used instead of the standard acryla- mide. For CE-SSCP analysis, data suggest that a sen- sitivity of 98 – 99% can be obtained using a 10% long chain poly- N , N -dimethylacrylamide polymer (LPDMA; Jespersgaard et al. , 2006 ). 2. Buffer composition . So far, the Tris-borate buffer is the buffer of choice for most investigators in SSCP analysis. However, in certain instances HEPES buffer has been demonstrated to offer an alternative solu- tion that may increase the sensitivity of the SSCP. The addition of 5% glycerol has been shown to lower the pH and to decrease the electrostatic repulsion between the negatively charged phosphates in the nucleic acid backbone, resulting in a higher resolution between the mutant and wild-type DNA fragments. Conformational structures can also be more compacted by increasing the salt concentration. Finally, buffer systems with low pH have been shown to increase the sensitivity for mutation detection in larger DNA fragments ( Kukita et al. , 1997 ). 3. Gel length and duration of gel run . There is a consid- erable variation in the duration of the gel run that has been adopted by the various diagnostic laboratories. It is inevitable that the time of electrophoresis is depend- ent on both the length of the gel as well as the applied voltage. It is preferable to start the electrophoresis with a relatively moderate voltage and increase it as soon as the PCR fragments have been migrated into the gel. Chapter | 4 Mutation Detection by Single Strand Conformation Polymorphism and Heteroduplex Analysis 51 the SSCP detection method based on statistical arguments has been developed. This method is based on the fact that the chance of any given strand to exhibit a mobility shift is independent from the other strand. On this assumption, the probability of observing shifts in both strands (P2), in one strand (P1), or in none of the strands (P0), is equal to x 2 , 2 x (1  x ) and (1  x ) 2 , respectively, where x is the sensitiv- ity of the technique when only one of the strands is labeled. The ratio of P2 to P1 is equal to the observed number of the mutations on both strands over the number of the observed mutations on the single strand [ r  ( x /2(1  x )]. The useful sensitivity of the technique can be calculated as being equal to 1  P0. Based on this probabilistic theory, the estimates from previous studies are that the sensitivity for 100 – 200 bp fragments is approximately 96%, regardless of the pres- ence or absence of glycerol ( Hayashi and Yandell, 1993 ). However, by adding glycerol, the sensitivity is still high for fragments ranging from 200 to 300 bp, but is decreas- ing when glycerol is not used. The latter may indicate the inability of the former calculation to depict the actual elec- trophoretic mobility, which to some extent is expected. In addition, it may also explain the fact that substitutions, which induce significant conformational changes, are most likely to have an effect, to a variable extent, on the other strand. Despite this discrepancy, it can be safely concluded that decreasing the fragment size will greatly enhance the sensitivity. It seems rational that the overall effect of a given mutation is displayed more efficiently when the total number of nucleotides surrounding that particular mutation is less. It is also profound that glycerol greatly increases the overall sensitivity when electrophoresis is performed at room temperature. Practical observations suggest that any fragment that exhibits a differential mobility will often migrate very close to the reference fragment. However, the overall frag- ment number is not always predictable in advance. Any given number of conformations may be supported to a variable extent by the applied electrophoretic parameters. Furthermore, the band intensity is irrelevant to allelic dif- ferences, as it is strictly dependent on the different confor- mations. Therefore, SSCP is not a safe method to predict gene-dosage effects. In addition, the simultaneous detec- tion of more than one mutation in a single DNA sample is not easily predictable, as previous data have shown that the electrophoretic pattern may vary considerably within differ- ent experiments. In general, although highly reproducible, the mobility of single-stranded DNA conformers cannot be predicted in advance from sequence information. Such attempts to predict SSCP mobility changes by modeling single-stranded DNA conformations using the structure prediction program Mfold ( http://frontend.bioinfo.rpi.edu/ applications/mfold/ ) have not provided consistent results. Improvements in modeling the structures of single-stranded DNA would make it possible to more accurately predicting SSCP mobility ( Nakabayashi and Nishigaki, 1996 ), the idiosyncratic nature of SSCP remains its main weakness as a diagnostic tool ( Liu et al. , 1999 ). Attempts to further improve the sensitivity of the SSCP have led to the development of the RNA-SSCP approach ( Sarkar et al. , 1992 ). Here, although the method is essen- tially the same, the double-stranded DNA is converted to the corresponding single-stranded RNA by means of one of the two primers that has phage promoter sequences on its 5  end. This method has shown a higher sensitivity com- pared to the previously described SSCP methodology. Its sensitivity may rely on the fact that the in vitro transcribed strand has no complementary strand to reanneal with. Therefore, sufficient amounts of the in vitro transcribed product can be electrophoresed and easily analyzed even by ethidium bromide staining ( Sarkar et al. , 1992 ). So far, there has been much discussion concerning the false negative results of this assay and possible ways to minimize them. However, false positive results also affect the net outcome of the SSCP analysis. In order to mini- mize the frequency of reporting false positive results, it is advisable to perform repeated SSCP electrophoretic runs (particularly when SSCP data are of clinical interest). An additional way is to determine the minimum mobility vari- ation, which is detectable within the context of laboratory SSCP conditions. In practical terms, mobility differences of 3 mm are generally clear, but a detection difference of only 2 mm requires excellent gel running conditions and is often subjective. Therefore, any difference smaller than or equal to 2 mm can be considered only with reservation. Reproducibility is the last and perhaps most essential parameter applicable to most techniques in molecular diag- nostics. In general, if conditions are kept constant then the resulting reproducibility is usually high. Nevertheless, Hayashi (1991) has previously suggested that DNA sequences may have different stable conformations. The latter is thus interpreted as a variable that may compromise SSCP repro- ducibility. It relies on the possibility that when the free energy difference between different conformations is small, an oscil- lation between different structures of comparable energies may be observed. 4.6.2 Sensitivity of HDA So far, the sensitivity of the HDA methodology has not been determined to the extent of the SSCP analysis. Rossetti and colleagues (1995) compared directly both SSCP and HDA assays for the detection of known mutations in a panel of four genes. Despite the fact that none of the assays was per- formed with 100% efficiency, HDA detected slightly more mutations than SSCP in the same samples. Interestingly, these authors suggested that both techniques could be used in concert to detect all mutations. Another aspect to improve the performance of HDA is the gel matrix. As in SSCP, the use of MDE (derived from HydroLink D5000 ™ ; Keen et al. , 1991 ) has basically made Molecular Diagnostics52 HDA a valuable mutation detection technique. Today, the majority of the diagnostic laboratories, in which HDA is the method of choice to detect genomic variation, are employing MDE gels. Finally , as already mentioned in section 4.5, the bubble- type heteroduplexes, which are formed due to the presence of single base substitutions, are much more difficult to visu- alize compared to the bulge-type heteroduplexes. In order to overcome this bottleneck, and based on the observa- tion that heteroduplexes are much easier to visualize when a deletion or insertion mutation is involved, the Universal Heteroduplex Generator (UHG) was conceived ( Wood et al. , 1993a, b ). In brief, the UHG consists of a synthetic DNA fragment, which bears a small (that is, 2 to 5 bp) dele- tion. This synthetic fragment is amplified by the use of the same oligonucleotide primers as the DNA under study. After amplification, the test amplicon is mixed with the amplified UHG, denatured, and then slowly reannealed, followed by electrophoresis. If no mutation is present in the test DNA, then only a bulge-type heteroduplex will be present, slightly retarded compared to the homoduplex. If, however, a single base substitution is also present, then the resulting heterodu- plex will have two mismatches: a bulge and a bubble type, which will result in the heteroduplex migrating significantly lower, compared to the simple bulge-type heteroduplex. The use of UHG has been reported for the detection of known mutations within a number of loci, such as von Willebrand disease ( Wood et al. , 1995 ), phenylketonurea ( Wood et al. , 1993a ), and for prenatal determination of blood group alle- les ( Stoerker et al. , 1996 ). 4.7 DETECTION OF THE UNDERLYING GENOMIC VARIATION USING SSCP AND HDA Due to their numerous advantages, SSCP and HDA anal- yses are nowadays the methods of choice in a growing number of private or public molecular diagnostic labo- ratories to either interrogate known mutations or scan for known or unknown mutations in short stretches of DNA and in relatively short time. Similarly, SSCP and HDA have also been proven to be invaluable tools for basic sci- ence, enabling both the identification of causative genes for human hereditary diseases and mapping of genomic loci. A short summary of the existing applications of SSCP and HDA follows in the next paragraphs, which is only indicative for the applicability of these techniques in almost every genomic locus. 4.7.1 Applications in Basic Science The utilization of SSCP in basic science as a tool for genomic DNA analysis is well established. Mutations in sev- eral key candidate genes implicated in various cell processes have now been identified, and the extent of those mutations as well as their frequency has given an insight into the role of these molecules in the relevant processes. Damage to DNA is considered to be the main initiating event by which genotoxins cause hereditary effects and can- cer. An accumulation of mutations throughout the genome will eventually result in cell death or in a cascade of events, which in turn may initiate malignant transformation. Therefore, it is not surprising that SSCP analysis was first used in screening candidate genes in tumorigenesis ( Suzuki et al. , 1990 ; Yap and McGee, 1992b ). Since then, several tissue specimens have been examined and nearly all possi- ble tumor types, isolated from a variety of tissue resources, have been considered for mutations in several key suspect genes. A difficulty often was the fact that many genes such as the RB or BRCA1 and BRCA2 genes have an enormous size, comprising several exons, and their analysis was often cumbersome if not impossible. Nevertheless, a number of research groups, including ours, have identified a multitude of p53 and nm-23 genomic alterations in almost every tumor type, such as in breast, colorectal (see Fig. 4.5 ), prostate, ovarian, and so on. In effect, this methodology proved to be FIGURE 4.5 Typical examples of silver stained SSCP gels, in which different genomic loci, responsible for tumorigenesis, are analyzed. A. Analysis of the nm-23 gene. Lanes A, D, and G correspond to three different heterozygous cases; lanes B, C, E, and F are normal individuals (Garinis, G. and Menounos, P.G., unpublished). B. Analysis of the human p53 locus. Different electrophoretic mobility of both the single-stranded (SS) and double-stranded (DS) alleles, due to a small deletion (depicted by an asterisk) in a heterozygous breast cancer affected individual (mt), compared to the wild-type (wt) electrophoretic pattern (adapted from Patrinos et al. , 1999 , with permission). C. Analysis of the human HBE (  -globin) gene. Lanes A and B: Heterozygote and homozygous cases for the presence of the  /HincII polymorphism respectively. Lanes C, D: Homozygous cases for the absence of the  /HincII polymorphism. (Adapted from Papachatzopoulou et al. , 2006 .) Chapter | 4 Mutation Detection by Single Strand Conformation Polymorphism and Heteroduplex Analysis 53 particularly useful in revealing, in a stepwise approach, that the altered expression of several cell cycle regulatory mol- ecules either at the genomic or transcriptional and protein levels may exert a synergetic effect on tumor growth and chromosomal instability on breast cancer and non-small cell lung and colorectal carcinomas. In addition to the utilization of SSCP methodology as a screening tool, several investigators have previously employed this technique for gene mapping in mouse genes ( Beier, 1993 and references therein). The methodology is based on the fact that a given polymorphism readily can be found in non-coding regions of genes such as the 3  untrans- lated regions or introns, between alleles of mouse species, and in several occasions between inbred strains as well. The segregations of these polymorphisms can be analyzed with recombinant inbred or interspecific crosses and the strain distribution pattern obtained can be compared with that for other markers and analyzed by standard linkage analy- sis algorithms. For instance, SSCP has previously been employed to localize 39 mouse-specific sequence-tagged sites (STSs), generated from mouse – hamster somatic cell hybrids. These were subsequently integrated with other markers to generate a high-density map of mouse chro- mosome 1 containing over 100 markers typed on a single interspecific backcross ( Watson et al. , 1992 ). SSCP analy- sis also has been applied in relatively fewer cases for the linkage analysis of human genes ( Nishimura et al. , 1993 ; Avramopoulos et al. , 1993 ). 4.7.2 Molecular Diagnostic Applications Both conventional and fluorescent or capillary-based SSCP and HDA can be successfully used for the detection of known mutations in any genomic locus. A brief summary of the numerous applications of SSCP analysis for various human genes is given in Table 4.1 . When SSCP analysis is coupled to non-radioactive detection schemes, then it most certainly becomes the method of choice for routine molec- ular diagnostic analysis (see Fig. 4.6 for representative examples from G6PD mutation screening ( Menounos et al. , TABLE 4.1 Summary of the majority of genomic loci, for which an SSCP mutation analysis strategy is designed and implemented. Genes Disease/syndrome References Ras TP53 Various types of cancer Suzuki et al . 1990 Sheffield et al . (1993) , Kutach et al. (1999) , Makino et al. (2000) * BRCA1 BRCA2 Susceptibility to breast cancer Castilla et al. (1994) Phelan et al. (1996) RB1 Retinoblastoma Hogg et al. (1992) , Shimizu et al. (1994) APC Adenomatous polyposis coli Groden et al. (1993) , Varesco et al. (1993) CYP21 Congenital adrenal hyperplasia Bobba et al . (1997) PAH Phenylketonurea Dockhorn-Dworniczak et al. (1991) , Labrune et al. (1991) G6PD Glycose-6-dehydrogenase deficiency Calabr ò et al. (1993) HFE Hereditary hemochromatosis Hertzberg et al. (1998) F7 Hemophilia A Economou et al. (1992) F9 Hemophilia B David et al. (1993) SPTA1 SPTB Hereditary elliptocytosis and spherocytosis Maillet et al. (1996) ANK1 SLC4A1 (Band-3) Hereditary spherocytosis Eber et al. (1996) Jarolim et al. (1996) HBA2, HBA1 α -thalassemia Harteveld et al. (1996) (Continued) Molecular Diagnostics56 heterogeneity of glucose-6-phosphate dehydrogenase deficiency rev- ealed by single-strand conformation and sequence analysis . Am. J. Hum. Genet. 52 , 527 – 536 . Castilla , L.H. , Couch , F.J. , Erdos , M.R. , Hoskins , K.F. , Calzone , K. , Garber , J.E. , Boyd , J. , Lubin , M.B. , Deshano , M.L. , Brody , L.C. , Collins , F.S. , and Weber , B.L. ( 1994 ) . Mutations in the BRCA1 gene in families with early-onset breast and ovarian cancer . Nat. Genet. 8 , 387 – 391 . Chehab , F.F. , and Kan , Y.W. ( 1989 ) . Detection of specific DNA sequences by fluorescence amplification: a color complementation assay . Proc. Natl. Acad. Sci. USA 86 , 9178 – 9182 . Clarkson , P.A. , Davies , H.R. , Williams , D.M. , Chaudhary , R. , Hughes , I.A. , and Patterson , M.N. ( 1993 ) . Mutational screening of the Wilms’s tumour gene, WT1, in males with genital abnormalities . J. Med. Genet. 30 , 767 – 772 . Claustres , M. , Laussel , M. , Desgeorges , M. , Giansily , M. , Culard , J.F. , Razakatsara , G. , and Demaille , J. ( 1993 ) . Analysis of the 27 exons and flanking regions of the cystic fibrosis gene: 40 different mutations account for 91.2% of the mutant alleles in southern France . Hum. Mol. Genet. 2 , 1209 – 1213 . David , D. , Rosa , H.A. , Pemberton , S. , Diniz , M.J. , Campos , M. , and Lavinha , J. ( 1993 ) . Single-strand conformation polymorphism (SSCP) analysis of the molecular pathology of hemophilia B . Hum. Mutat. 2 , 355 – 361 . Day , I.N. , Whittall , R. , Gudnason , V. , and Humphries , S.E. ( 1995 ) . Dried tem- plate DNA, dried PCR oligonucleotides and mailing in 96-well plates: LDL receptor gene mutation screening . Biotechniques 18 , 981 – 984 . Dobson-Stone , C. , Cox , R.D. , Lonie , L. , Southam , L. , Fraser , M. , Wise , C. , Bernier , F. , Hodgson , S. , Porter , D.E. , Simpson , A.H. , and Monaco , A.P. ( 2000 ) . Comparison of fluorescent single-strand conformation polymorphism analysis and denaturing high-performance liquid chro- matography for detection of EXT1 and EXT2 mutations in hereditary multiple exostoses . Eur. J. Hum. Genet. 8 , 24 – 32 . Dockhorn-Dworniczak , B. , Dworniczak , B. , Brommelkamp , L. , Bulles , J. , Horst , J. , and Bocker , W.W. ( 1991 ) . Non-isotopic detection of single strand conformation polymorphism (PCRSSCP): a rapid and sensi- tive technique in diagnosis of phenylketonuria . Nucleic Acids Res. 19 , 2500 . Dryja , T.P. , Hahn , L.B. , Cowley , G.S. , McGee , T.L. , and Berson , E.L. ( 1991 ) . Mutation spectrum of the rhodopsin gene among patients with autosomal dominant retinitis pigmentosa . Proc. Natl. Acad. Sci. USA 88 , 9370 – 9374 . Eber , S.W. , Gonzalez , J.M. , Lux , M.L. , Scarpa , A.L. , Tse , W.T. , Dornwell , M. , Herbers , J. , Kugler , W. , Ozcan , R. , Pekrun , A. , Gallagher , P.G. , Schroter , W. , Forget , B.G. , and Lux , S.E. ( 1996 ) . Ankyrin-1 mutations are a major cause of dominant and recessive hereditary spherocytosis . Nat. Genet. 13 , 214 – 218 . Economou , E.P. , Kazazian , H.H. , Jr. , and Antonarakis , S.E. ( 1992 ) . Detection of mutations in the factor VIII gene using single-stranded conformational polymorphism (SSCP) . Genomics 13 , 909 – 911 . Ellis, L.A., Taylor, C.F., and Taylor, G.R. (2000). A comparison of fluo- rescent SSCP and denaturing HPCL for high throughput mutation scanning. Hum. Mutat. 15, 556 – 564. Ellison , J.S. ( 1996 ) . Fluorescence-based mutation detection. Single-strand conformation polymorphism analysis (F-SSCP) . Mol. Biotechnol. 5 , 17 – 31 . Ellison , J. , Dean , M. , and Goldman , D. ( 1993 ) . Efficacy of fluorescence- based PCR-SSCP for detection of point mutation . BioTechniques 15 , 684 – 691 . Esteban-Carde ñ osa , E. , Duran , M. , Infante , M. , Velasco , E. , and Miner , C. ( 2004 ) . High-throughput mutation detection method to scan BRCA1 and BRCA2 based on heteroduplex analysis by capillary array elec- trophoresis . Clin. Chem. 50 , 313 – 320 . Groden , J. , Gelbert , L. , Thliveris , A. , Nelson , L. , Robertson , M. , Joslyn , G. , Samowitz , W. , Spirio , L. , Carlson , M. , Burt , R. , Leppert , M. , and White , R. ( 1993 ) . Mutational analysis of patients with adenomatous polyposis: identical inactivating mutations in unrelated individuals . Am. J. Hum. Genet. 52 , 263 – 272 . Harteveld , K.L. , Heister , A.J. , Giordano , P.C. , Losekoot , M. , and Bernini , L.F. ( 1996 ) . Rapid detection of point mutations and polymor- phisms of the alpha-globin genes by DGGE and SSCA . Hum. Mutat. 7 , 114 – 122 . Hayashi , K. , and Yandell , D.W. ( 1993 ) . How sensitive is PCRSSCP? Hum. Mutat. 2 , 338 – 346 . Hayashi , K. ( 1991 ) . PCR-SSCP: a simple and sensitive method for detec- tion of mutations in the genomic DNA . PCR Methods Appl. 1 , 34 – 38 . Hayward , C. , Rae , A.L. , Porteous , M.E. , Logie , L.J. , and Brock , D.J. ( 1994 ) . Two novel mutations and a neutral polymorphism in EGF-like domains of the fibrillin gene (FBN1): SSCP screening of exons 15 – 21 in Marfan syndrome patients . Hum. Mol. Genet. 3 , 373 – 375 . Hertzberg , M.S. , McDonald , D. , and Mirochnik , O. ( 1998 ) . Rapid diagno- sis of hemochromatosis gene Cys282Tyr mutation by SSCP analysis . Am. J. Hematol. 57 , 260 – 261 . Hogg , A. , Onadim , Z. , Baird , P.N. , and Cowell , J.K. ( 1992 ) . Detection of heterozygous mutations in the RB1 gene in retinoblastoma patients using single-strand conformation polymorphism analysis and polymer- ase chain reaction sequencing . Oncogene 7 , 1445 – 1451 . Humphries , S.E. , Gudnason , V. , Whittall , R. , and Day , I.N. ( 1997 ) . Single- strand conformation polymorphism analysis with high throughput modifications, and its use in mutation detection in familial hypercho- lesterolemia. International Federation of Clinical Chemistry Scientific Division: Committee on Molecular Biology Techniques . Clin. Chem. 43 , 427 – 435 . Inazuka , M. , Wenz , H.M. , Sakabe , M. , Tahira , T. , and Hayashi , K. ( 1997 ) . A streamlined mutation detection system: multicolor post-PCR fluo- rescence labeling and single-strand conformational polymorphism analysis by capillary electrophoresis . Genome Res. 7 , 1094 – 1103 . Iwahana , H. , Yoshimoto , K. , Mizusawa , N. , Kudo , E. , and Itakura , M. ( 1994 ) . Multiple fluorescence-based PCR-SSCP analysis . Bio- techniques 16 , 296 – 305 . Jarolim , P. , Murray , J.L. , Rubin , H.L. , Taylor , W.M. , Prchal , J.T. , Ballas , S.K. , Snyder , L.M. , Chrobak , L. , Melrose , W.D. , Brabec , V. , and Palek , J. ( 1996 ) . Characterization of 13 novel band 3 gene defects in hereditary spherocytosis with band 3 deficiency . Blood 88 , 4366 – 4374 . Jespersgaard , C. , Larsen , L.A. , Baba , S. , Kukita , Y. , Tahira , T. , Christiansen , M. , Vuust , J. , Hayashi , K. , and Andersen , P.S. ( 2006 ) . Optimization of capillary array electrophoresis single-strand con- formation polymorphism analysis for routine molecular diagnostics . Electrophoresis 27 , 3816 – 3822 . Kawame , H. , Hasegawa , Y. , Eto , Y. , and Maekawa , K. ( 1992 ) . Rapid identification of mutations in the glucocerebrosidase gene of Gaucher disease patients by analysis of single-strand conformation polymor- phisms . Hum. Genet. 90 , 294 – 296 . Keen , J. , Lester , D. , Inglehearn , C. , Curtis , A. , and Bhattacharya , S. ( 1991 ) . Rapid detection of single base mismatches as heteroduplexes on Hydrolink gels . Trends Genet. 7 , 5 . Kozlowski , P. , and Krzyzosiak , W.J. ( 2001 ) . Combined SSCP/duplex anal- ysis by capillary electrophoresis for more efficient mutation detection . Nucleic Acids Res. 29 , E71 . Kukita , Y. , Tahira , T. , Sommer , S.S. , and Hayashi , K. ( 1997 ) . SSCP analy- sis of long DNA fragments in low pH gel . Hum. Mutat. 10 , 400 – 407 . Chapter | 4 Mutation Detection by Single Strand Conformation Polymorphism and Heteroduplex Analysis 57 Kutach , L.S. , Bolshakov , S. , and Ananthaswamy , H.N. ( 1999 ) . Detection of mutations and polymorphisms in the p53 tumor suppressor gene by single-strand conformation polymorphism analysis . Electrophoresis 20 , 1204 – 1210 . Labrune , P. , Melle , D. , Rey , F. , Berthelon , M. , Caillaud , C. , Rey , J. , Munnich , A. , and Lyonnet , S. ( 1991 ) . Single-strand conformation polymorphism for detection of mutations and base substitutions in phenylketonuria . Am. J. Hum. Genet. 48 , 1115 – 1120 . L á zaro , C. , and Estivill , X. ( 1992 ) . Mutation analysis of genetic dis- eases by asymmetric-PCR SSCP and ethidium bromide staining: application to neurofibromatosis and cystic fibrosis . Mol. Cell. Probes 6 , 357 – 359 . Levran , O. , Erlich , T. , Magdalena , N. , Gregory , J.J. , Batish , S.D. , Verlander , P.C. , and Auerbach , A.D. ( 1997 ) . Sequence variation in the Fanconi anemia gene FAA . Proc. Natl. Acad. Sci. USA 94 , 13051 – 13056 . Liu , Q. , Feng , J. , Buzin , C. , Wen , C. , Nozari , G. , Mengos , A. , Nguyen , V. , Liu , J. , Crawford , L. , Fujimura , F.K. , and Sommer , S.S. ( 1999 ) . Detection of virtually all mutations-SSCP (DOVAM-S): a rapid method for mutation scanning with virtually 100% sensitivity . Biotechniques 26 , 932 . Liu , X.Z. , Xia , X.J. , Adams , J. , Chen , Z.Y. , Welch , K.O. , Tekin , M. , Ouyang , X.M. , Kristiansen , A. , Pandya , A. , Balkany , T. , Arnos , K.S. , and Nance , W.E. ( 2001 ) . Mutations in GJA1 (connexin 43) are asso- ciated with non-syndromic autosomal recessive deafness . Hum. Mol. Genet. 10 , 2945 – 2951 . MacCollin , M. , Ramesh , V. , Jacoby , L.B. , Louis , D.N. , Rubio , M.P. , Pulaski , K. , Trofatter , J.A. , Short , M.P. , Bove , C. , Eldridge , R. , Parry , D.M. , and Gusella , J.F. ( 1994 ) . Mutational analysis of patients with neurofibromatosis 2 . Am. J. Hum. Genet. 55 , 314 – 320 . Maillet , P. , Alloisio , N. , Morle , L. , and Delaunay , J. ( 1996 ) . Spectrin mutations in hereditary elliptocytosis and hereditary spherocytosis . Hum. Mutat. 8 , 97 – 107 . Makino , R. , Yazyu , H. , Kishimoto , Y. , Sekiya , T. , and Hayashi , K. ( 1992 ) . F-SSCP: fluorescence-based polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis . PCR Methods Applic. 2 , 10 – 13 . Makino , R. , Kaneko , K. , Kurahashi , T. , Matsumura , T. , and Mitamura , K. ( 2000 ) . Detection of mutation of the p53 gene with high sensitivity by fluorescence-based PCR-SSCP analysis using low-pH buffer and an automated DNA sequencer in a large number of DNA samples . Mutat. Res. 452 , 83 – 90 . Medintz , I. , Wong , W.W. , Sensabaugh , G. , and Mathies , R.A. ( 2000 ) . High speed single nucleotide polymorphism typing of a hereditary haemochromatosis mutation with capillary array electrophoresis microplates . Electrophoresis 21 , 2352 – 2358 . Medintz , I.L. , Paegel , B.M. , Blazej , R.G. , Emrich , C.A. , Berti , L. , Scherer , J.R. , and Mathies , R.A. ( 2001 ) . High-performance genetic analysis using micro- fabricated capillary array electrophoresis microplates . Electrophoresis 22 , 3845 – 3856 . Menounos , P. , Zervas , C. , Garinis , G. , Doukas , C. , Kolokithopoulos , D. , Tegos , C. , and Patrinos , G.P. ( 2000 ) . Molecular heterogeneity of the glucose-6-phosphate dehydrogenase deficiency in the Hellenic popu- lation . Hum. Hered. 50 , 237 – 241 . Myers , R.M. , Maniatis , T. , and Lerman , L.S. ( 1987 ) . Detection and locali- zation of single base changes by denaturing gradient gel electrophore- sis . Methods Enzymol. 155 , 501 – 527 . Nair , S. , Lin , T.K. , Pang , T. , and Altwegg , M. ( 2002 ) . Characterization of Salmonella serovars by PCR-single-strand conformation polymor- phism analysis . J. Clin. Microbiol. 40 , 2346 – 2351 . Nakabayashi , Y. , and Nishigaki , K. ( 1996 ) . Single-strand conformation polymorphism (SSCP) can be explained by semistable conformation dynamics of single-stranded DNA . J. Biochem. (Tokyo) 120 , 320 – 325 . Nishimura , D.Y. , Purchio , A.F. , and Murray , J.C. ( 1993 ) . Linkage locali- zation of TGFB2 and the human homeobox gene HLX1 to chromo- some 1q . Genomics 15 , 357 – 364 . Orita , M. , Iwahana , H. , Kanazawa , H. , Hayashi , K. , and Sekiya , T. ( 1989 ) . Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms . Proc. Natl. Acad. Sci. USA 86 , 2766 – 2770 . Papachatzopoulou , A. , Menounos , P.G. , Kolonelou , C. , and Patrinos , G.P. ( 2006 ) . Mutation screening in the human epsilon-globin gene using single-strand conformation polymorphism analysis . Am. J. Hematol. 81 , 136 – 138 . Patrinos , G.P. , Garinis , G. , Kounelis , S. , Kouri , E. , and Menounos , P. ( 1999 ) . A novel 23-bp deletion in exon 5 of the p53 tumor suppressor gene . J. Mol. Med. 77 , 686 – 689 . Paul , P. , Letteboer , T. , Gelbert , L. , Groden , J. , White , R. , and Coppes , M.J. ( 1993 ) . Identical APC exon 15 mutations result in a variable phenotype in familial adenomatous polyposis . Hum. Mol. Genet. 2 , 925 – 931 . Peters , T. , Schlayer , H.J. , Hiller , B. , R ö sler , B. , Blum , H. , and Rasenack , J. ( 1997 ) . Quasispecies analysis in hepatitis C virus infection by fluores- cent single strand conformation polymorphism . J. Virol. Methods. 64 , 95 – 102 . Phelan , C.M. , Lancaster , J.M. , Tonin , P. , Gumbs , C. , Cochran , C. , Carter , R. , Ghadirian , P. , Perret , C. , Moslehi , R. , Dion , F. , Faucher , M.C. , Dole , K. , Karimi , S. , Foulkes , W. , Lounis , H. , Warner , E. , Goss , P. , Anderson , D. , Larsson , C. , Narod , S.A. , and Futreal , P.A. ( 1996 ) . Mutation analysis of the BRCA2 gene in 49 site-specific breast cancer families . Nat. Genet. 13 , 120 – 122 . Quintanar , A. , Jallu , V. , Legros , Y. , and Kaplan , C. ( 1998 ) . Human platelet antigen genotyping using a fluorescent SSCP technique with an auto- matic sequencer . Br. J. Haematol. 103 , 437 – 444 . Ravnik-Glavac , M. , Glavac , D. , and Dean , M. ( 1994 ) . Sensitivity of single-strand conformation polymorphism and heteroduplex method for mutation detection in the cystic fibrosis gene . Hum. Mol. Genet. 3 , 801 – 807 . Rommens , J. , Kerem , B.S. , Greer , W. , Chang , P. , Tsui , L.C. , and Ray , P. ( 1990 ) . Rapid nonradioactive detection of the major cystic fibrosis mutation . Am. J. Hum. Genet. 46 , 395 – 396 . Rossetti , S. , Corra , S. , Biasi , M.O. , Turco , A.E. , and Pignatti , P.F. ( 1995 ) . Comparison of heteroduplex and single-strand conformation analyses, followed by ethidium fluorescence visualization, for the detection of mutations in four human genes . Mol. Cell. Probes 9 , 195 – 200 . Saeki , Y. , Ueno , S. , Takahashi , N. , Soga , F. , and Yanagihara , T. ( 1992 ) . A novel mutant (transthyretin Ile-50) related to amyloid polyneuropa- thy. Single-strand conformation polymorphism as a new genetic marker . FEBS Lett. 308 , 35 – 37 . Sainz , J. , Huynh , D.P. , Figueroa , K. , Ragge , N.K. , Baser , M.E. , and Pulst , S.M. ( 1994 ) . Mutations of the neurofibromatosis gene and lack of the gene product in vestibular schwannomas . Hum. Mol. Genet. 3 , 885 – 891 . Sarkar , G. , Yoon , H.S. , and Sommer , S.S. ( 1992 ) . Screening for mutations by RNA single-strand conformation polymorphism (rSSCP): compar- ison with DNA-SSCP . Nucleic Acids Res. 20 , 871 – 878 . Scott , D.A. , Kraft , M.L. , Carmi , R. , Ramesh , A. , Elbedour , K. , Yairi , Y. , Srisailapathy , C.R. , Rosengren , S.S. , Markham , A.F. , Mueller , R.F. , Lench , N.J. , Van Camp , G. , Smith , R.J. , and Sheffield , V.C. ( 1998 ) . Identification of mutations in the connexin 26 gene that cause auto- somal recessive nonsyndromic hearing loss . Hum. Mutat. 11 , 387 – 394 . Molecular Diagnostics58 Sheffield , V.C. , Beck , J.S. , Kwitek , A.E. , Sandstrom , D.W. , and Stone , E.M. ( 1993 ) . The sensitivity of single-strand conformation polymorphism anal- ysis for the detection of single base substitutions . Genomics 16 , 325 – 332 . Shen , M.H. , Harper , P.S. , and Upadhyaya , M. ( 1993 ) . Neurofibromatosis type 1 (NF1): the search for mutations by PCR heteroduplex analysis on Hydrolink gels . Hum. Mol. Genet. 2 , 1861 – 1864 . Shimizu , T. , Toguchida , J. , Kato , M.V. , Kaneko , A. , Ishizaki , K. , and Sasaki , M.S. ( 1994 ) . Detection of mutations of the RB1 gene in retin- oblastoma patients by using exon-by-exon PCRSSCP analysis . Am. J. Hum. Genet. 54 , 793 – 800 . S ö zen , M.M. , Whittall , R. , Oner , C. , Tokatli , A. , Kalkanoğlu , H.S. , Dursun , A. , Coşkun , T. , Oner , R. , and Humphries , S.E. ( 2005 ) . The molecular basis of familial hypercholesterolaemia in Turkish patients . Atherosclerosis 180 , 63 – 71 . Stoerker , J. , Hurwitz , C. , Rose , N.C. , Silberstein , L.E. , and Highsmith , W.E. ( 1996 ) . Heteroduplex generator in analysis of Rh blood group alleles . Clin. Chem. 42 , 356 – 360 . Strom , T.M. , Hortnagel , K. , Hofmann , S. , Gekeler , F. , Scharfe , C. , Rabl , W. , Gerbitz , K.D. , and Meitinger , T. ( 1998 ) . Diabetes insipidus, diabetes mellitus, optic atrophy and deafness (DIDMOAD) caused by mutations in a novel gene (wolframin) coding for a predicted transmembrane pro- tein . Hum. Mol. Genet. 7 , 2021 – 2028 . Suzuki , Y. , Orita , M. , Shiraishi , M. , Hayashi , K. , and Sekiya , T. ( 1990 ) . Detection of ras gene mutations in human lung cancers by single- strand conformation polymorphism analysis of polymerase chain reaction products . Oncogene 5 , 1037 – 1043 . Takahashi-Fujii , A. , Ishino , Y. , Kato , I. , and Fukumaki , Y. ( 1994 ) . Rapid and practical detection of beta-globin mutations causing beta-tha- lassemia by fluorescence-based PCR-single-stranded conformation polymorphism analysis . Mol. Cell. Probes 8 , 385 – 393 . Thomson, K.L., Gloyn, A.L., Colclough, K., Batten, M., Allen, L.I. Beards, F., Hattersley, A.T., and Ellard, S. (2003). Identification of 21 novel glucokinase (GCK) mutations in UK and European Caucasians with maturity-onset diabetes of the young (MODY). Hum. Mutat. 22, 417. Tuffery , S. , Moine , P. , Demaille , J. , and Claustres , M. ( 1993 ) . Base substi- tutions in the human dystrophin gene: detection by using the single- strand conformation polymorphism (SSCP) technique . Hum. Mutat. 2 , 368 – 374 . Upadhyaya , M. , Maynard , J. , Osborn , M. , Huson , S.M. , Ponder , M. , Ponder , B.A. , and Harper , P.S. ( 1995 ) . Characterisation of germline mutations in the neurofibromatosis type 1 (NF1) gene . J. Med. Genet. 32 , 706 – 710 . Varesco , L. , Gismondi , V. , James , R. , Robertson , M. , Grammatico , P. , Groden , J. , Casarino , L. , De Benedetti , L. , Bafico , A. , Bertario , L. , Sala , P. , Sassatelli , R. , Ponz de Leon , M. , Biasco , G. , Allergetti , A. , Aste , H. , De Sanctis , S. , Rossetti , C. , Illeni , M.T. , Sciarra , A. , Del Porto , G. , White , R. , and Ferrara , G.B. ( 1993 ) . Identification of APC gene mutations in Italian adenomatous polyposis coli patients by PCR-SSCP analysis . Am. J. Hum. Genet. 52 , 280 – 285 . Veldhuisen , B. , Saris , J.J. , de Haij , S. , Hayashi , T. , Reynolds , D.M. , Mochizuki , T. , Elles , R. , Fossdal , R. , Bogdanova , N. , van Dijk , M.A. , Coto , E. , Ravine , D. , Norby , S. , Verellen-Dumoulin , C. , Breuning , M.H. , Somlo , S. , and Peters , D.J. ( 1997 ) . A spectrum of mutations in the sec- ond gene for autosomal dominant polycystic kidney disease (PKD2) . Am. J. Hum. Genet. 61 , 547 – 555 . Wang , Y.H. , Barker , P. , and Griffith , J. ( 1992 ) . Visualization of diagnos- tic heteroduplex DNAs from cystic fibrosis deletion heterozygotes provides an estimate of the kinking of DNA by bulged bases . J. Biol. Chem. 267 , 4911 – 4915 . Watson , M.L. , D’Eustachio , P. , Mock , B.A. , Steinberg , A.D. , Morse , H.C. , 3rd , Oakey , R.J. , Howard , T.A. , Rochelle , J.M. , and Seldin , M.F. ( 1992 ) . A linkage map of mouse chromosome 1 using an interspecific cross segregating for the gld autoimmunity mutation . Mamm. Genome 2 , 158 – 171 . White , M.B. , Carvalho , M. , Derse , D. , O’Brien , S.J. , and Dean , M. ( 1992 ) . Detecting single base substitutions as heteroduplex polymorphisms . Genomics 12 , 301 – 306 . Whittall , R. , Gudnason , V. , Weavind , G.P. , Day , L.B. , Humphries , S.E. , and Day , I.N. ( 1995 ) . Utilities for high throughput use of the single strand conformational polymorphism method: screening of 791 patients with familial hypercholesterolaemia for mutations in exon 3 of the low den- sity lipoprotein receptor gene . J. Med. Genet. 32 , 509 – 515 . Wood , N. , Tyfield , L. , and Bidwell , J. ( 1993 a ) . Rapid classification of phenylketonuria genotypes by analysis of heteroduplexes generated by PCR-amplifiable synthetic DNA . Hum. Mutat. 2 , 131 – 137 . Wood , N. , Standen , G. , Hows , J. , Bradley , B. , and Bidwell , J. ( 1993 b ) . Diagnosis of sickle-cell disease with a universal heteroduplex genera- tor . Lancet 342 , 1519 – 1520 . Wood , N. , Standen , G.R. , Murray , E.W. , Lillicrap , D. , Holmberg , L. , Peake , I.R. , and Bidwell , J. ( 1995 ) . Rapid genotype analysis in type 2B von Willebrand’s disease using a universal heteroduplex generator . Br. J. Haematol. 89 , 152 – 156 . Yap , E.P. , and McGee , J.O. ( 1992 a ) . Nonisotopic SSCP detection in PCR products by ethidium bromide staining . Trends Genet. 8 , 49 . Yap , E.P. , and McGee , J.O. ( 1992 b ) . Nonisotopic SSCP and competitive PCR for DNA quantification: p53 in breast cancer cells . Nucleic Acids Res. 20 , 145 . Zappella , M. , Meloni , I. , Longo , I. , Canitano , R. , Hayek , G. , Rosaia , L. , Mari , F. , and Renieri , A. ( 2003 ) . Study of MECP2 gene in Rett syn- drome variants and autistic girls . Am. J. Med. Genet. 119B , 102 – 107 . Chapter | 5 Capillary Electrophoresis 61 5.3 CAPILLARY ELECTROPHORESIS IN MOLECULAR DIAGNOSTICS Nucleotides have been quantified in different matrices, including tissue and cell extracts and several DNA and RNA sources. Therapeutic antisense oligonucleotides are of inter- est for many applications and CE can be used to character- ize and quantify these materials ( Righetti and Gelfi, 1998 ). DNA separations are generally conducted by CGE, in which gel- or polymer-filled capillaries act as sieving media to resolve the different lengths of DNA. DNA of dif- ferent lengths has similar electrophoretic mobility as each increase in size is accompanied by a corresponding increase in the number of negative charges. Therefore, the separa- tion by mobility differences is not the preferred approach and the majority of separations are achieved using a sieve mechanism. The capillary is filled with a matrix of synthetic or natural polymer. The various DNA fragments migrate through this matrix and become entangled with or trapped in the matrix. The migration of the larger DNA fragment is retarded to a greater extent, which results in a size-based separation mechanism. A variety of compounds, such as bis-polyacrylamide, agarose, or methylcellulose, have been used to act as a molecular sieve to cause separation ( Ren, 2000 ). One of the most active research areas for compound separation has been the investigation of alternative sieving matrices to replace gels ( Cottet et al. , 1998 ; Magnusdottir et al. , 1998 ). Recently, a nanostructured copolymer matrix has been suc- cessfully used to separate oligonucleotides with high reso- lution by CE using a very short separation channel which simulates real microchip conditions ( Zhang et al. , 2006 ). Generally , it is difficult to prepare gel-filled capillaries manually due to bubble formation. Further, gels have a lim- ited lifetime, since they are easily destroyed by high current and joule heating. CE requires a liquid medium, and the best choice appears to be a semi-dilute solution of polymers, which is very much similar to gel, since the molecules are entan- gled with one another in a way that they create a fine mesh ( Sunada and Blanch, 1997 ). Entangled polymer solutions (ESCE, entangled polymer solutions CE) can be replaced after each run and are therefore much easier to operate. Further, capillary non-gel sieving electrophoresis (CNGSE) has been employed in the biological sciences for the size-based separa- tion of macromolecules such as nucleic acids and facilitates the application of CE for detection of mutations and polymor- phisms in human molecular diagnostics. 5.4 MODES OF APPLICATION Besides gene sequencing most protocols adapted to automated CE represent analyses of DNA fragment length or DNA restriction patterns (RFLP), analyses of single-strand con- formation polymorphism (SSCP, see also previous chapter), and microsatellite analyses. 5.4.1 Sequencing Sequence analysis is the gold standard for molecular genetic testing. It yields the greatest amount of information since it identifies the order of each deoxynucleotide base of a par- ticular target, usually amplified DNA or cDNA. Although PCR techniques made sequencing routine, detection and analysis of the resulting DNA fragments became the major bottleneck for large-scale DNA sequencing ( Dovichi, 1997 ; Schmalzing et al. , 1999 ). Although the identification of the human genome sequence is completed, the importance of DNA sequencing has not been diminished. Sequencing of other organisms ’ genomes, comparative genomics or screening for human genetic defects are still challenging fields of application for the future. DNA sequencing using dye-labeled didesoxynucleotides and the method developed by Sanger and coworkers (1992) were adapted to CE. The single-base resolving capability of CE permits all four fluorochrome products to be sepa- rated simultaneously compared to classical sequencing with PAGE, where each chain terminator has to be loaded in sepa- rate lanes. The result is a multicolored ladder where each color represents a different base. The order of the bases is then analyzed using secondary software that characterizes the sequence in terms of identity, and relatedness to prototypical sequences in a database. Today CE serves as the routine high- throughput technique for sequencing. CE systems with multi- array capillaries ( Tan and Yeung, 1998 ; Wu et al. , 2008 ; Zhang et al. , 1999 ) will further enhance high throughput. 5.4.2 Analyses of DNA Fragment Length or Restriction Patterns and Microsatellites Cleavage of DNA products and analysis of the DNA cleav- age pattern by CE is currently widely used to detect gene mutations ( Andersen et al. , 1998 ; Sell and Lugemwa, 1999 ). The method is applicable in cases where, due to mutations or polymorphisms, restriction sites are lost or gained. In contrast to sequencing, fragment analysis incorpo- rates one fluorochrome-labeled PCR primer in a stand- ard PCR reaction after which the product(s) are separated and visualized. Fragment analysis is more rapid and less expensive because the fluorochrome is incorporated in the initial PCR reaction thus eliminating the need for sequenc- ing reactions that incorporate the dye terminating bases. Interpretation of the fragment analysis data is also easier than sequence analysis because the fragments are readily identified by the imaging software and the need for second- ary sequence software analysis is not required. Using this technique, some research groups established protocols for high-speed separation that allow distinguish- ing between wild-type and mutant PCR products extremely rapidly ( Chan et al. , 1996 ) with a short effective length capillary and high field strength. While less expensive and more rapid, fragment analysis does not provide exact, detailed sequence data. In addition, Molecular Diagnostics62 PCR fragments can result from erroneous amplification and thus verification using sequencing techniques or independ- ent probes is sometimes recommended for quality assur- ance purposes. In these cases, sequencing is often used to confirm the identity of the PCR product. Because of problems due to incomplete cleavage the method is being replaced by SSCP where possible. Furthermore , microsatellite analysis was originally per- formed using gel electrophoresis and radioactively labeled PCR products of defined areas of the genome. Application on CE made this method faster and more reliable (see para- graph below). 5.4.3 Analyses of Single-Strand Conformation Polymorphism Genetic diagnosis of an inherited disease or cancer predis- position often involves search for unknown point mutations in several genes. SSCP, a method exploiting visualization of different secondary structures under native conditions combined with CE, was shown by many groups to be a rapid and automated technique for processing large num- bers of samples (see also previous chapter). Larsen and coworkers (1999) used the method to detect point muta- tions associated with the inherited cardiac disorders long QT syndrome (LQTS) and hypertrophic cardiomyopathy (HCM). Sensitivity has been reported to be almost 100%, when 34 different point mutations were analyzed, while 10 previously unknown variants were found. These results clearly demonstrate that the method has high resolution, good reproducibility, and is very robust. Based on the pos- sibility of automation and short time of analysis, the method should be suitable for high-throughput applications such as genetic screening of large populations. Several other groups have already established this method for specific screening of mutations in other diseases (see below). 5.4.4 Other Applications CDCE has been combined with high-fidelity DNA ampli- fication and automated multicapillary instrument with fraction collection allowing sensitive, automated, and cost- effective analysis of healthy human tissues and population screening for disease-associated single nucleotide polymor- phisms (SNPs) in large pooled samples ( Muniappan and Thilly, 1999 ; Li et al. , 2005 ). Further applications of CDCE include studies of somatic mitochondrial mutations with respect to aging and measurement of mutational spectra of nuclear genes. The technique of CDCE was also shown to be applicable for detection of mutations in the K-ras and N-ras genes ( Kumar et al. , 1995 ; Bjorheim et al. , 1998 ). Kuypers and coworkers (1996) developed a method for online melting of double-stranded DNA for SSCP-CE, while they further improved the integration of PCR and CE by composing a contamination-free, automated PCR in the CE apparatus ( Kuypers et al. , 1998 ). Further , RT-PCR followed by CE had been used to quantify the expression levels of specific gene products ( Borson et al. , 1998 ; Butler, 1998 ; Odin et al. , 1999 ). Bor and coworkers (2000) described a protocol for simultane- ous quantification of several mRNA species, after cali- brating RT-PCR with CE. In a study of Schummer and coworkers (1999) , the clinical relevance of multidrug resistance-associated protein (MRP) gene expression was correlated with chemoresistance in prostate carcinoma. De Cremoux and coworkers (1999) compared the c-erbB-2 gene amplification in breast cancer with the expression of c-erbB-2 protein evaluated by immunohistochemistry and found a concordance of 91% between the two techniques. They concluded that c-erbB-2 gene amplification can be accurately quantitated by competitive PCR followed by CE and that this method is also suitable for small, fixed tissue specimens. Stanta and Bonin (1998) also used CE to quan- tify specific RT-PCR products and were able to show that the quantitation by CE is more reliable than by dot-blot. Finally , Kuypers and coworkers (1994) compared the use of CE with that of slab gel electrophoresis for quantifi- cation of chromosomal translocations in lymphoma, show- ing that the results of both methods are comparable. 5.5 SPECIFIC DIAGNOSTIC APPLICATIONS 5.5.1 Diagnosis of Neoplastic Disorders In the diagnosis of neoplastic disorders, CE has been used for the detection of chromosomal aberrations, microsatel- lite instability, clonality assays, and the detection of several other cancer relevant genetic mutations, like SNPs. Colorectal cancer is the leading cause of death related to cancer in western countries ( Weir et al. , 2003 ). Molecular biology studies have led to the identification of two broad categories of molecular alterations in colorectal cancer. Loss of heterozygosity (LOH) represents 80% of colorectal can- cers and is characterized by aneuploidy or allelic losses. The second group displays phenotypic microsatellite instability (MSI-positive tumors), has a near-diploid karyotype and a relatively low frequency of allelic losses. It accounts for 15% of all colorectal cancers. As a consequence of these two phe- nomena, other specific genetic events occur at high frequency. These include inactivation of tumor suppressor genes by genetic (deletion or mutation) or epigenetic events ( Garinis et al. , 2002 ), activation of proto-oncogenes by mutation, and dysregulated expression of several other molecules known to be involved in the development of colorectal cancer. 5.5.1.1 Loss of Heterozygosity Aneuploidy indicates gross losses or gains in chromosomal DNA and is often seen in many human primary tumors and Chapter | 5 Capillary Electrophoresis 63 premalignant conditions. Loss of one allele at a chromo- somal locus may imply the presence of a tumor suppres- sor gene at that site. Loss of both alleles at a given locus (homozygous deletion) is an even stronger indicator of the existence of a tumor suppressor gene. Many of these loci are already associated with one or more known candidate tumor suppressor genes, including 17p ( p53 gene), 5q21 ( APC gene), 3p21 ( β -catenin ( CTNNB1 ) gene), 9p ( p16 and p15 genes), and 13q ( RB gene). LOH analysis was originally performed using gel electrophoresis and radioactively labeled PCR products of defined areas to the genome. The use of CE made this analysis more reliable and faster ( Canzian et al. , 1996 ). CE was used for the detection of the loss of several tumor sup- pressor genes in micro-dissected cancerous tissue ( Marsh et al. , 2003 ). Gene fragments were amplified using PCR with flanking oligonucleotides bearing fluorescent labels and subsequently separated and analyzed by CE. The p53 gene locus is the commonest site demonstrat- ing loss of heterozygosity. TP53 is a DNA binding protein, which is a transcriptional activator and can cause cell cycle arrest in response to DNA damage. A second tumor suppressor gene adenomatous polyposis coli ( APC ) is inactivated in more than 80% of early colorec- tal cancers. An important function of the APC gene is to pre- vent the accumulation of molecules associated with cancer, such as catenins. Familial adenomatous polyposis (FAP) is an autosomal dominant disease caused by germline mutations in the APC gene. FAP is a rare condition in which hundreds or thousands of polyps develop along the length of the colon, and, if left untreated, lead to colon cancer. Figure 5.2 depicts LOH of the APC locus in tumor compared to normal tissue. An additional application of LOH assays is the identi- fication of chromosomal differences between normal and tumor tissue. This is useful in distinguishing between tumor recurrence vs. de novo cancer formation ( Rolston et al. , 2001 ; Sasatomi et al. , 2002 ). 5.5.1.2 Microsatellite Instability Microsatellite instability (MSI), or replication error, com- prises length alterations of oligonucleotide repeat sequences that occur somatically in human tumors. They are the man- ifestation of genomic instability where tumor cells have a decreased overall ability to faithfully replicate DNA. MSI is a frequent, if not obligatory, surrogate marker of underlying functional inactivation of one of the human DNA mismatch repair genes ( MLH1 , MSH2 , MSH6 , PMS1 , PMS2 (reviewed in Jacob and Praz, 2002 )). DNA mismatch repair enzymes normally remove misincor- porated single or multiple nucleotide bases as a result of random errors during recombination or replications. Functional loss of mismatch repair occurs due to biallelic inactivation via combination of gene mutation, LOH and/or promoter methylation ( Herman et al. , 1998 ; Jacob and Praz, 2002 ). Germline mutation of a mismatch repair gene has been shown to be the autosomal dominant genetic defect in most hereditary non-polyposis colorectal cancer (HNPCC) patients ( Marra and Boland, 1995 ; Saletti et al. , 2001 ). A second hit incurred in tumor cells in HNPCC individ- uals results in biallelic inactivation of the specific MMR gene. This results in loss of faithful replication of mic- rosatellite DNA in tumor ( Saletti et al. , 2001 ). Bethesda criteria ( Henson et al. , 1995 ) have been outlined to guide the identification of this syndrome. However, finding MSI positive tumors was shown to be the best predictor of germline mutation ( Liu et al. , 1999 ). Although implicating a germline defect in HNPCC patients, MSI is also found in 15 – 20% of sporadic colon cancer ( Goel et al. , 2003 ), where the finding reflects an overall increase in genomic instability. CE has been used for microsatellite analysis as well as for sequencing of DNA mismatch repair genes. Figure 5.3 illustrates mutation detection in the human MSH-2 gene, using CE-based DNA sequencing, while Fig. 5.4 depicts the result of the analysis of two marker regions of MSI, comparing normal and tumor tissue. Many different microsatellite markers and loci have been used to identify MSI in tumors. In 1997, the US National Cancer Institute (NCI) recommended a panel of microsatellite markers for use in colorectal cancer MSI testing, and the continued use of the MSI marker panel is still recommended ( Boland et al. , 1998 ; Muller et al. , 2004 ). Berg and coworkers (2000) described a fluorescent multiplex PCR-capillary electrophoresis (FM-CE) assay that permits the simultaneous detection of all five loci pro- posed by the NCI. Further, a protocol for SSCP-CE screen- ing for alterations in the exon of the MSH2 gene leading to the possibility of high-throughput screening has been published ( Merkelbach-Bruse et al., 2000 ). This protocol significantly reduces time and expenses, compared to con- ventional sequence determination. FIGURE 5.2 Analysis for LOH by CE. Genomic DNA is isolated from normal or tumor tissue, amplified with specific primers for defined chro- mosomal loci and the PCR products are subsequently separated by CE. The two alleles are represented by two peaks in DNA isolated from nor- mal tissue, automatically marked with gray color by the analysis software. In contrast, in DNA from the tumor tissue, one peak is missing (arrow). Molecular Diagnostics66 5,000 males. Almost all cases are caused by expansion of a (CGG) n trinucleotide repeat within the 5  untranslated region of the FMR1 (fragile X mental retardation) gene transcript. Until today the disease was reliably diagnosed by Southern blotting, requiring large samples and high input of time. Larsen and coworkers (1997) employed automated CE for accurate and high-throughput analysis of the FRAXA (CGG) n region in the normal and permutation range. Their method is based on PCR amplification of extracted genomic DNA followed by automated CE and detection of mul- ticolor fluorescence. The method proved to be useful in both research and clinical mutation screening when a large number of samples, predominantly in the normal range of amplification, are to be analyzed. Most recently, Strom and coworkers (2007) developed a new method called capillary Southern analysis that allows automated high-throughput screening for FMR1 alleles. Initially, samples are analyzed by a multiplex PCR that contains an internal control to establish gender. Only females homozygous at the FMR1 locus are further analyzed by capillary Southern analysis. Theoretically, this method can detect expansion as high as 2,000 CGG repeats, although Strom and coworkers (2007) found the largest non-mosaic FMR1 present in their series was 950 CGG repeats. Also , Huntington disease (HD) belongs to the group of neurodegenerative disorders characterized by unstable expanded trinucleotide repeats. In the case of HD the expan- sion of a CAG repeat occurs in the IT15 gene. Williams and colleagues have established CE analysis for sizing CAG repeats and showed that it enables confident use in sizing HD alleles ( Williams et al. , 1999 ). Further applications of CE involve multi-allelic-specific amplification in the analysis of the 21-hydroxylase gene for patients with congenital adrenal hyperplasia ( Barta et al. , 2001 ), and prenatal diagnosis of β -thalassemia, one of the most common recessive inherited disorders where many different mutations need to be detected ( Trent et al. , 1998 ). Recently, a chip-based capillary electrophoresis detecting system was described that facilitates the rapid and sensitive prenatal diagnosis of β -thalassemia ( Hu et al. , 2008 ). Geisel and coworkers (1999) described an SSCP-CE method to screen for unknown mutations in the low-density lipoprotein ( LDL ) receptor gene. They PCR-amplified the promoter region as well as all 18 exons and tested the accu- racy of the developed technique by reproducing 61 known genetic variations by a distinct abnormal SSCP pattern. The c.C677T mutation of the methylentetrahydrofolate reductase ( MTHFR ) gene is a nutrient-oriented mutation that is associated with elevated levels of homocysteine and an increased risk of coronary heart disease . An opti- mized assay for automated PCR-RFLP genotyping of the MTHFR gene was established by Sell and Lugemwa (1999) . Following amplification, the resulting PCR prod- uct was digested by the HinfI restriction enzonuclease, and the resulting fragments were analyzed by CE. The method was shown to be suitable for high-throughput screening and will support the screening of large sample sizes. Saffroy and coworkers (2002) introduced a multiplex analysis of mutations in factor V Leiden (c.G1691A), prothrombin (c.G20210A), and 5,10-methylenetetrahydrofolate reductase (c.C677T), which have been associated with an enhanced risk of thrombosis, using a CE setting. This technique can be applied to specimens from large clinical trials and epide- miological surveys. Hereditary hemochromatosis (HH) represents an auto- somal recessive disease in which increased iron absorption causes iron overload and irreversible tissue damage. Two point mutations in the HFE gene on chromosome 6p have been found to be associated with HH and led to the possibil- ity of patient screening before the onset of irreversible tissue damage. Jackson and coworkers (1997) have developed a heteroduplex analysis using capillary electrophoresis for the detection of the p.C282Y mutation. An SSCP-CE approach has been recently adapted for the detection of point muta- tions in codon 63 or 282 of HH patients ( Bosserhoff et al. , 1999 ), indicating that SSCP-CE is a reliable, cost-effective, sensitive, and rapid method for genotyping HFE mutations ( Fig. 5.6 ). Nevertheless, CE can be performed equally well for RFLP analysis in order to provide diagnosis of HFE gene mutations ( Fig. 5.7 ). The hereditary Charcot Marie Tooth 1 A neuropathy (CMT1A) and hereditary neuropathy with liability to pres- sure palsies (HNPP) are caused by a duplication and a deletion, respectively, at chromosome 17p11.2 – p12 encom- passing the peripheral myelin protein 22 ( PMP22 ) gene. Multiplex PCR followed by capillary electrophoresis pro- vides a rapid and reliable detection system for duplications/ deletions of the PMP22 gene ( Lin et al. , 2006 ). Finally , several commercially available kits have been developed for the diagnosis of mutations in the cystic fibrosis gene applying CE base techniques ( Tomaiuolo et al. , 2003 ). One is based on multiplex oligonucleotide ligation assay and allows the screening of 31 different mutations in the CFTR gene, corresponding to approximately 95% of the mutated CFTR alleles ( Zielenski et al. , 2002 ). 5.5.3 Diagnosis of Infectious Diseases Diagnosis of infectious diseases is a fast growing field for CE applications. In general, molecular methods do not require the presence of viable organisms permitting the identification of bacteria, viruses, and fungi that are difficult if not impos- sible in culture. Molecular identification of infectious agents can be used for both diagnostic and therapeutic purposes and CE has the main advantages of including higher throughput and sensitivity than conventional methods. Chronic hepatitis C virus (HCV) infection is a world- wide public health problem with a global prevalence of 2%. Attallah and coworkers (2004) proved that CZE provides Chapter | 5 Capillary Electrophoresis 67 a rapid and inexpensive method for diagnosis and mass screening of a large number of HCV-infected individuals. Further, since it has been shown that different HCV geno- types are associated with distinct profiles of pathogenicity and responses to antiviral treatment, demand for HCV geno- typing has increased. Doglio and coworkers (1998) devel- oped a CE-based detection mode, which in combination with direct cycle sequencing provides a simple and rapid method for routine HCV genotyping. More recently, temperature gradient capillary electrophoresis (TGCE) was introduced as a rapid and inexpensive method for genotyping of HCV that does not require sequencing ( Margraf et al. , 2004 ). Kolesar and coworkers (1997) used CE coupled to a laser- induced fluorescence technique (CE-LIF) to directly quantify HIV-1 RNA. They developed a fluorescent-labeled DNA probe with optimal stability and sensitivity for RNA hybridi- zation of HIV-1 RNA isolated from plasma and showed that as little as 19fg of HIV RNA could be reliably and quantita- tively detected. The technique of enterobacterial repetitive intergenic con- sensus (ERIC)-PCR produces genomic DNA fingerprints that allow discrimination between bacterial species and strains. Sciacchitano (1998) applied this technique coupled to CE to differentiate Listeria monocytogenes , an important food- borne pathogen implicated in numerous cases of listeriosis. Hernandez and coworkers (1999) developed a scheme for rapid identification of Mycobacterium sp. based on a combined restriction enzyme-CE analysis of PCR-amplified DNA and showed this detection method to be com- parable to conventional methods for identification of mycobacteria. SSCP -CE was used by Ghozzi and coworkers (1999) to rapidly identify Pseudomonas aeruginosa and other gram- negative non-fermenting bacilli from patients with cystic fibrosis. These authors have shown that this approach is suited for rapid identification of the main gram-negative non-fermenting bacteria. In addition to diagnosis of infectious diseases, CE-based techniques such as micellar electrokinetic chromatography are used for the analysis of antibiotics and antiviral drugs. 5.5.4 Identity Testing and Forensic Applications In 1985, multi- and single-locus DNA probes for the detection of RFLPs were first applied to identify specific individuals. Later, the introduction of PCR for specific amplification of short tandem repeat (STR) polymorphisms (microsatellites) represented a major breakthrough, and STR typing methods are widely used today for human identity testing including forensic DNA analysis. Following multiplex PCR amplification, DNA samples containing the length-variant STR alleles are typically separated by cap- illary electrophoresis and genotyped by comparison to an allelic ladder ( Butler, 2007 ). Currently , 6 – 16 fluorescently labeled STR loci are ana- lyzed simultaneously with a single PCR amplification. As part of multiplex PCR kits, sex determination of forensic samples can be obtained using CE-based analysis of the X – Y homologous gene amelogenin ( La Fountain et al. , 1998 ; Pouchkarev et al. , 1998 ). The analysis of 6 – 10 STRs FIGURE 5.6 Analysis of codon 282 of the HFE gene in HH patients with a combined SSCP-CE analysis. Three typical SSCP profiles of codon 282 PCR fragments are shown. Panel A. Shows the analysis of a normal individual. Panel B. Displays the result of a patient with the mutation at codon 282 of the HFE gene in the homogyzous state (one peak at a differ- ent position than in the wild-type sequence). In panel C. A heterozygous genotype is seen (two peaks). Together with the PCR products analyzed, the HD-400-ROX standard was applied, clearly allowing the identification of the characteristic peaks of the individual genotypes. Molecular Diagnostics68 FIGURE 5.7 RFLP analysis using CE. RFLP analysis of genomic DNA by classical gel electrophoresis can be replaced by CE as mentioned in the text. PCR reaction and digestion of the PCR product is performed following the standard protocol but using one fluorescent-labeled primer. Subsequent anal- ysis by CE reveals characteristic patterns for the individual genotypes. As an example, analysis of the HFE gene is shown. The HFE mutation at codon 282 generates a new restriction site. Digestion with the according restriction enzyme results in two shorter DNA fragments (only the one with the align- ing fluorescent-labeled primer at one end can be detected by CE). In panels A. and B. Only one peak at different positions is seen, corresponding to the wild-type (digested) and mutated sequence of the HFE gene (undigested), respectively. In panel C. The heterozygous genotype is displayed (two peaks). (Courtesy of A. Hartmann, University of Regensburg, Germany.) Chapter | 5 Capillary Electrophoresis 71 expression of the matrix metalloproteinase-1 gene in ovarian cancers and an insertion/deletion polymorphism in its promoter region . Cancer Res. 59 , 4225 – 4227 . Kandioler , D. , Zwrtek , R. , Ludwig , C. , Janschek , E. , Ploner , M. , Hofbauer , F. , Kuhrer , I. , Kappel , S. , Wrba , F. , Horvath , M. , Karner , J. , Renner , K. , Bergmann , M. , Karner-Hanusch , J. , Potter , R. , Jakesz , R. , Teleky , B. , and Herbst , F. ( 2002 ) . TP53 genotype but not p53 immunohistochemical result predicts response to preoperative short-term radiotherapy in rectal cancer . Ann. Surg. 235 , 493 – 498 . Khrapko , K. , Hanekamp , J.S. , Thilly , W.G. , Belenkii , A. , Foret , F. , and Karger , B.L. ( 1994 ) . Constant denaturant capillary electrophoresis (CDCE): a high resolution approach to mutational analysis . Nucleic Acids Res. 22 , 364 – 369 . Kolesar , J.M. , Allen , P.G. , and Doran , C.M. ( 1997 ) . Direct quantification of HIV-1 RNA by capillary electrophoresis with laser-induced fluo- rescence . J. Chromatogr. B. Biomed Sci. Appl. 697 , 189 – 194 . Kumar , R. , Hanekamp , J.S. , Louhelainen , J. , Burvall , K. , Onfelt , A. , Hemminki , K. , and Thilly , W.G. ( 1995 ) . Separation of transforming amino acid-substituting mutations in codons 12, 13 and 61 the N- ras gene by constant denaturant capillary electrophoresis (CDCE) . Carcinogenesis 16 , 2667 – 2673 . Kuypers , A.W. , Willems , P.M. , van der Schans , M.J. , Linssen , P.C. , Wessels , H.M. , de Bruijn , C.H. , Everaerts , F.M. , and Mensink , E.J. ( 1993 ) . Detection of point mutations in DNA using capillary electro- phoresis in a polymer network . J. Chromatogr. 621 , 149 – 156 . Kuypers , A.W. , Meijerink , J.P. , Smetsers , T.F. , Linssen , P.C. , and Mensink , E.J. ( 1994 ) . Quantitative analysis of DNA aberrations amplified by competitive polymerase chain reaction using capillary electrophoresis . J. Chromatogr. B. Biomed Appl. 660 , 271 – 277 . Kuypers , A.W. , Linssen , P.C. , Willems , P.M. , and Mensink , E.J. ( 1996 ) . On-line melting of double-stranded DNA for analysis of single- stranded DNA using capillary electrophoresis . J. Chromatogr. B. Biomed Appl. 675 , 205 – 211 . Kuypers , A.W. , Linssen , P.C. , Lauer , H.H. , and Mensink , E.J. ( 1998 ) . Contamination-free and automated composition of a reaction mixture for nucleic acid amplification using a capillary electrophoresis appa- ratus . J. Chromatogr. A. 806 , 141 – 147 . La Fountain , M. , Schwartz , M. , Cormier , J. , and Buel , E. ( 1998 ) . Validation of capillary electrophoresis for analysis of the X – Y homol- ogous amelogenin gene . J. Forensic Sci. 43 , 1188 – 1194 . Lander , E.S. , Linton , L.M. , Birren , B. , Nusbaum , C. , Zody , M.C. , Baldwin , J. , Devon , K. , Dewar , K. , Doyle , M. , FitzHugh , W. , Funke , R. , Gage , D. , Harris , K. , Heaford , A. , Howland , J. , Kann , L. , Lehoczky , J. , LeVine , R. , McEwan , P. , McKernan , K. , Meldrim , J. , Mesirov , J.P. , Miranda , C. , Morris , W. , Naylor , J. , Raymond , C. , Rosetti , M. , Santos , R. , Sheridan , A. , Sougnez , C. , Stange-Thomann , N. , Stojanovic , N. , Subramanian , A. , Wyman , D. , Rogers , J. , Sulston , J. , Ainscough , R. , Beck , S. , Bentley , D. , Burton , J. , Clee , C. , Carter , N. , Coulson , A. , Deadman , R. , Deloukas , P. , Dunham , A. , Dunham , I. , Durbin , R. , French , L. , Grafham , D. , Gregory , S. , Hubbard , T. , Humphray , S. , Hunt , A. , Jones , M. , Lloyd , C. , McMurray , A. , Matthews , L. , Mercer , S. , Milne , S. , Mullikin , J.C. , Mungall , A. , Plumb , R. , Ross , M. , Shownkeen , R. , Sims , S. , Waterston , R.H. , Wilson , R.K. , Hillier , L.W. , McPherson , L.W. , Marra , M.A. , Mardis , E.R. , Fulton , L.A. , Chinwalla , A.T. , Pepin , K.H. , Gish , W.R. , Chissoe , S.L. , Wendl , M.C. , Delehaunty , K.D. , Miner , T.L. , Delehaunty , A. , Kramer , J.B. , Cook , L.L. , Fulton , R.S. , Johnson , D.L. , Minx , P.J. , Clifton , S.W. , Hawkins , T. , Branscomb , E. , Predki , P. , Richardson , P. , Wenning , S. , Slezak , T. , Doggett , N. , Cheng , J.F. , Olsen , A. , Lucas , S. , Elkin , C. , Uberbacher , E. , Frazier , M. , Gibbs , R.A. , Muzny , D.M. , Scherer , S.E. , Bouck , J.B. , Sodergren , E.J. , Worley , K.C. , Rives , C.M. , Gorrell , J.H. , Metzker , M.L. , Naylor , S.L. , Kucherlapati , R.S. , Nelson , D.L. , Weinstock , G.M. , Sakaki , Y. , Fujiyama , A. , Hattori , M. , Yada , T. , Toyoda , A. , Itoh , T. , Kawagoe , C. , Watanabe , H. , Totoki , Y. , Taylor , T. , Weissenbach , J. , Heilig , R. , Saurin , W. , Artiguenave , F. , Brottier , P. , Bruls , T. , Pelletier , E. , Robert , C. , Wincker , P. , Smith , D.R. , Doucette-Stamm , L. , Rubenfield , M. , Weinstock , M. , Lee , H.M. , Dubois , J. , Rosenthal , A. , Platzer , M. , Nyakatura , G. , Taudien , S. , Rump , A. , Yang , H. , Yu , J. , Wang , J. , Huang , G. , Gu , J. , Hood , L. , Rowen , L. , Madan , A. , Qin , S. , Davis , R.W. , Federspiel , N.A. , Abola , A.P. , Proctor , M.J. , Myers , R.M. , Schmutz , J. , Dickson , M. , Grimwood , J. , Cox , D.R. , Olson , M.V. , Kaul , R. , Raymond , C. , Shimizu , N. , Kawasaki , K. , Minoshima , S. , Evans , G.A. , Athanasiou , M. , Schultz , R. , Roe , B.A. , Chen , F. , Pan , H. , Ramser , J. , Lehrach , H. , Reinhardt , R. , McCombie , W.R. , de la N Dedhia , B.M. , Blocker , H. , Hornischer , K. , Nordsiek , G. , Agarwala , R. , Aravind , L. , Bailey , J.A. , Bateman , A. , Batzoglou , S. , Birney , E. , Bork , P. , Brown , D.G. , Burge , C.B. , Cerutti , L. , Chen , H.C. , Church , D. , Clamp , M. , Copley , R.R. , Doerks , T. , Eddy , S.R. , Eichler , E.E. , Furey , T.S. , Galagan , J. , Gilbert , J.G. , Harmon , C. , Hayashizaki , Y. , Haussler , D. , Hermjakob , H. , Hokamp , K. , Jang , W. , Johnson , L.S. , Jones , T.A. , Kasif , S. , Kaspryzk , A. , Kennedy , S. , Kent , W.J. , Kitts , P. , Koonin , E.V. , Korf , I. , Kulp , D. , Lancet , D. , Lowe , T.M. , McLysaght , A. , Mikkelsen , T. , Moran , J.V. , Mulder , N. , Pollara , V.J. , Ponting , C.P. , Schuler , G. , Schultz , J. , Slater , G. , Smit , A.F. , Stupka , E. , Szustakowski , J. , Thierry-Mieg , D. , Thierry-Mieg , J. , Wagner , L. , Wallis , J. , Wheeler , R. , Williams , A. , Wolf , Y.I. , Wolfe , K.H. , Yang , S.P. , Yeh , R.F. , Collins , F. , Guyer , M.S. , Peterson , J. , Felsenfeld , A. , Wetterstrand , K.A. , Patrinos , A. , Morgan , M.J. , Szustakowki , J. , de Jong , P. , Catanese , J.J. , Osoegawa , K. , Shizuya , H. , and Choi , S. ( 2001 ) . Initial sequencing and analysis of the human genome . Nature 409 , 860 – 921 . Larsen , L.A. , Gronskov , K. , Norgaard-Pedersen , B. , Brondum-Nielsen , K. , Hasholt , L. , and Vuust , J. ( 1997 ) . High-throughput analysis of fragile X (CGG)n alleles in the normal and premutation range by PCR amplifica- tion and automated capillary electrophoresis . Hum. Genet. 100 , 564 – 568 . Larsen , L.A. , Christiansen , M. , Vuust , J. , and Andersen , P.S. ( 1999 ) . High-throughput single-strand conformation polymorphism analy- sis by automated capillary electrophoresis: robust multiplex analysis and pattern-based identification of allelic variants . Hum. Mutat. 13 , 318 – 327 . Li-Sucholeiki , X.C. , Khrapko , K. , Andre , P.C. , Marcelino , L.A. , Karger , B.L. , and Thilly , W.G. ( 1999 ) . Applications of constant denaturant capil- lary electrophoresis/high-fidelity polymerase chain reaction to human genetic analysis . Electrophoresis 20 , 1224 – 1232 . Li , Q. , Deka , C. , Glassner , B.J. , Arnold , K. , Li-Sucholeiki , X.C. , Tomita- Mitchell , A. , Thilly , W.G. , and Karger , B.L. ( 2005 ) . Design of an automated multicapillary instrument with fraction collection for DNA mutation discovery by constant denaturant capillary electrophoresis (CDCE) . J. Sep. Sci. 28 , 1375 – 1389 . Lin , C.Y. , Su , Y.N. , Lee , C.N. , Hung , C.C. , Cheng , W.F. , Lin , W.L. , Chen , C.A. , and Hsieh , S.T. ( 2006 ) . A rapid and reliable detection system for the analysis of PMP22 gene dosage by MP/DHPLC assay . J. Hum. Genet. 51 , 227 – 235 . Liu , T. , Tannergard , P. , Hackman , P. , Rubio , C. , Kressner , U. , Lindmark , G. , Hellgren , D. , Lambert , B. , and Lindblom , A. ( 1999 ) . Missense muta- tions in hMLH1 associated with colorectal cancer . Hum. Genet. 105 , 437 – 441 . Lupski , J.R. , de Oca-Luna , R.M. , Slaugenhaupt , S. , Pentao , L. , Guzzetta , V. , Trask , B.J. , Saucedo-Cardenas , O. , Barker , D.F. , Killian , J.M. , and Garcia , C.A. ( 1991 ) . DNA duplication associated with Charcot- Marie-Tooth disease type 1 A . Cell 66 , 219 – 232 . Magnusdottir , S. , Viovy , J.L. , and Francois , J. ( 1998 ) . High resolution cap- illary electrophoretic separation of oligonucleotides in low-viscosity, hydrophobically end-capped polyethylene oxide with cubic order . Electrophoresis 19 , 1699 – 1703 . Molecular Diagnostics72 Mansfield , E.S. , Vainer , M. , Harris , D.W. , Gasparini , P. , Estivill , X. , Surrey , S. , and Fortina , P. ( 1997 ) . Rapid sizing of polymorphic mic- rosatellite markers by capillary array electrophoresis . J. Chromatogr. A. 781 , 295 – 305 . Marchi , E. , and Pasacreta , R.J. ( 1997 ) . Capillary electrophoresis in court: the landmark decision of the People of Tennessee versus Ware . J. Capillary Electrophor. 4 , 145 – 156 . Margraf , R.L. , Erali , M. , Liew , M. , and Wittwer , C.T. ( 2004 ) . Genotyping hepatitis C virus by heteroduplex mobility analysis using temperature gradient capillary electrophoresis . J. Clin. Microbiol. 42 , 4545 – 4551 . Marra , G. , and Boland , C.R. ( 1995 ) . Hereditary nonpolyposis colorectal cancer: the syndrome, the genes, and historical perspectives . J. Natl. Cancer Inst. 87 , 1114 – 1125 . Marsh , J.W. , Finkelstein , S.D. , Demetris , A.J. , Swalsky , P.A. , Sasatomi , E. , Bandos , A. , Subotin , M. , and Dvorchik , I. ( 2003 ) . Genotyping of hepatocellular carcinoma in liver transplant recipients adds predic- tive power for determining recurrence-free survival . Liver Transpl. 9 , 664 – 671 . Merkelbach-Bruse , S. , Kose , S. , Losen , I. , Bosserhoff , A.K. , and Buettner , R. ( 2000 ) . Detection of HNPCC patients by multiple use of capillary electro- phoresis techniques . Combin. Chem. High Throughput Screen 3 , 519 – 524 . Miettinen , M. , Majidi , M. , and Lasota , J. ( 2002 ) . Pathology and diagnos- tic criteria of gastrointestinal stromal tumors (GISTs): a review . Eur. J. Cancer 38 , S39 – S51 . Miller , J.E. , Wilson , S.S. , Jaye , D.L. , and Kronenberg , M. ( 1999 ) . An automated semiquantitative B and T cell clonality assay . Mol. Diagn. 4 , 101 – 117 . Muller , A. , Edmonston , T.B. , Dietmaier , W. , Buttner , R. , Fishel , R. , and Ruschoff , J. ( 2004 ) . MSI-testing in hereditary non-polyposis colorec- tal carcinoma (HNPCC) . Dis. Markers 20 , 225 – 236 . Muniappan , B.P. , and Thilly , W.G. ( 1999 ) . Application of constant denaturant capillary electrophoresis (CDCE) to mutation detection in humans . Genet. Anal. 14 , 221 – 227 . Munro , N.J. , Snow , K. , Kant , J.A. , and Landers , J.P. ( 1999 ) . Molecular diagnostics on microfabricated electrophoretic devices: from slab gel- to capillary- to microchip-based assays for T- and B-cell lymphopro- liferative disorders . Clin. Chem. 45 , 1906 – 1917 . Nicoletto , M.O. , Donach , M. , De Nicolo , A. , Artioli , G. , Banna , G. , and Monfardini , S. ( 2001 ) . BRCA-1 and BRCA-2 mutations as prognostic factors in clinical practice and genetic counseling . Cancer Treat. Rev. 27 , 295 – 304 . Odin , E. , Wettergren , Y. , Larsson , L. , Larsson , P.A. , and Gustavsson , B. ( 1999 ) . Rapid method for relative gene expression determination in human tissues using automated capillary gel electrophoresis and mul- ticolor detection . J. Chromatogr. B. Biomed Sci. Appl. 734 , 47 – 53 . Ordeig , O. , Godino , N. , del Campo , J. , Munoz , F.X. , Nikolajeff , F. , and Nyholm , L. ( 2008 ) . On-chip electric field driven electrochemical detection using a poly(dimethylsiloxane) microchannel with gold microband electrodes . Anal. Chem. 80 , 3622 – 3632 . Pancholi , P. , Oda , R.P. , Mitchell , P.S. , Landers , J.P. , and Persing , D.H. ( 1997 ) . Diagnostic detection of herpes simplex and Hepatitis C viral amplicons by Capillary Electrophoresis: Comparison with Southern blot detection . Mol. Diagn. 2 , 27 – 37 . Pouchkarev , V.P. , Shved , E.F. , and Novikov , P.I. ( 1998 ) . Sex determination of forensic samples by polymerase chain reaction of the amelogenin gene and analysis by capillary electrophoresis with polymer matrix . Electrophoresis 19 , 76 – 79 . Ren , J. ( 2000 ) . High-throughput single-strand conformation polymor- phism analysis by capillary electrophoresis . J. Chromatogr. B Biomed. Sci. Appl. 741 , 115 – 128 . Righetti , P.G. , and Gelfi , C. ( 1998 ) . Recent advances in capillary zone electrophoresis of DNA . Forensic Sci. Int. 92 , 239 – 250 . Rolston , R. , Sasatomi , E. , Hunt , J. , Swalsky , P.A. , and Finkelstein , S.D. ( 2001 ) . Distinguishing de novo second cancer formation from tumor recurrence: mutational fingerprinting by microdissection genotyping . J. Mol. Diagn. 3 , 129 – 132 . Saffroy , R. , Lemoine , A. , Haas , P. , Tindiliere , F. , Marion , S. , and Debuire , B. ( 2002 ) . Rapid automated simultaneous screening of (G1691A) Factor V, (G20210A) prothrombin, and (C677T) methylenetetrahy- drofolate reductase variants by multiplex PCR using fluorescence scanning technology . Genet. Test. 6 , 233 – 236 . Saletti , P. , Edwin , I.D. , Pack , K. , Cavalli , F. , and Atkin , W.S. ( 2001 ) . Microsatellite instability: application in hereditary non-polyposis colorectal cancer . Ann. Oncol. 12 , 151 – 160 . Sanger , F. , Nicklen , S. , and Coulson , A.R. ( 1992 ) . DNA sequencing with chain-terminating inhibitors. 1977 [classical article] . Biotechnology 24 , 104 – 108 . Sasatomi , E. , Finkelstein , S.D. , Woods , J.D. , Bakker , A. , Swalsky , P.A. , Luketich , J.D. , Fernando , H.C. , and Yousem , S.A. ( 2002 ) . Comparison of accumulated allele loss between primary tumor and lymph node metastasis in stage II non-small cell lung carcinoma: implications for the timing of lymph node metastasis and prognostic value . Cancer Res. 62 , 2681 – 2689 . Schmalzing , D. , Koutny , L. , Salas-Solano , O. , Adourian , A. , Matsudaira , P. , and Ehrlich , D. ( 1999 ) . Recent developments in DNA sequencing by cap- illary and microdevice electrophoresis . Electrophoresis 20 , 3066 – 3077 . Schummer , B. , Siegsmund , M. , Steidler , A. , Toktomambetova , L. , Kohrmann , K.U. , and Alken , P. ( 1999 ) . Expression of the gene for the multidrug resistance-associated protein in human prostate tissue . Urol. Res. 27 , 164 – 168 . Sciacchitano , C.J. ( 1998 ) . DNA fingerprinting of Listeria monocytogenes using enterobacterial repetitive intergenic consensus (ERIC) motifs- polymerase chain reaction/capillary electrophoresis . Electrophoresis 19 , 66 – 70 . Sell , S.M. , and Lugemwa , P.R. ( 1999 ) . Development of a highly accurate, rapid PCR-RFLP genotyping assay for the methylenetetrahydrofolate reductase gene . Genet. Test. 3 , 287 – 289 . Simpson , P.C. , Roach , D. , Woolley , A.T. , Thorsen , T. , Johnston , R. , Sensabaugh , G.F. , and Mathies , R.A. ( 1998 ) . High-throughput genetic analysis using microfabricated 96-sample capillary array electro- phoresis microplates . Proc. Natl. Acad. Sci. USA 95 , 2256 – 2261 . Stanta , G. , and Bonin , S. ( 1998 ) . RNA quantitative analysis from fixed and paraffin-embedded tissues: membrane hybridization and capillary electrophoresis . Biotechniques 24 , 271 – 276 . Strom , C.M. , Huang , D. , Li , Y. , Hantash , F.M. , Rooke , J. , Potts , S.J. , and Sun , W. ( 2007 ) . Development of a novel, accurate, automated, rapid, high-throughput technique suitable for population-based carrier screening for Fragile X syndrome . Genet. Med. 9 , 199 – 207 . Sunada , W.M. , and Blanch , H.W. ( 1997 ) . Polymeric separation media for cap- illary electrophoresis of nucleic acids . Electrophoresis 18 , 2243 – 2254 . Tan , H. , and Yeung , E.S. ( 1998 ) . Automation and integration of multi- plexed on-line sample preparation with capillary electrophoresis for high-throughput DNA sequencing . Anal. Chem. 70 , 4044 – 4053 . Terabe , S. ( 2004 ) . Micellar electrokinetic chromatography . Anal. Chem. 76 , 241A – 246A . Tomaiuolo , R. , Spina , M. , and Castaldo , G. ( 2003 ) . Molecular diagnosis of cystic fibrosis: comparison of four analytical procedures . Clin. Chem. Lab. Med. 41 , 26 – 32 . Trent , R.J. , Le , H. , and Yu , B. ( 1998 ) . Prenatal diagnosis for thalassaemia in a multicultural society . Prenat. Diagn. 18 , 591 – 598 . Chapter | 5 Capillary Electrophoresis 73 Tsui , L.C. ( 1992 a ) . Mutations and sequence variations detected in the cystic fibrosis transmembrane conductance regulator (CFTR) gene: a report from the Cystic Fibrosis Genetic Analysis Consortium . Hum. Mutat. 1 , 197 – 203 . Tsui , L.C. ( 1992 b ) . The spectrum of cystic fibrosis mutations . Trends Genet. 8 , 392 – 398 . Venter , J.C. , Adams , M.D. , Myers , E.W. , Li , P.W. , Mural , R.J. , Sutton , G.G. , Smith , H.O. , Yandell , M. , Evans , C.A. , Holt , R.A. , Gocayne , J.D. , Amanatides , P. , Ballew , R.M. , Huson , D.H. , Wortman , J.R. , Zhang , Q. , Kodira , C.D. , Zheng , X.H. , Chen , L. , Skupski , M. , Subramanian , G. , Thomas , P.D. , Zhang , J. , Gabor Miklos , G.L. , Nelson , C. , Broder , S. , Clark , A.G. , Nadeau , J. , McKusick , V.A. , Zinder , N. , Levine , A.J. , Roberts , R.J. , Simon , M. , Slayman , C. , Hunkapiller , M. , Bolanos , R. , Delcher , A. , Dew , I. , Fasulo , D. , Flanigan , M. , Florea , L. , Halpern , A. , Hannenhalli , S. , Kravitz , S. , Levy , S. , Mobarry , C. , Reinert , K. , Remington , K. , Abu-Threideh , J. , Beasley , E. , Biddick , K. , Bonazzi , V. , Brandon , R. , Cargill , M. , Chandramouliswaran , I. , Charlab , R. , Chaturvedi , K. , Deng , Z. , Di , F. , Dunn , P. , Eilbeck , K. , Evangelista , C. , Gabrielian , A.E. , Gan , W. , Ge , W. , Gong , F. , Gu , Z. , Guan , P. , Heiman , T.J. , Higgins , M.E. , Ji , R.R. , Ke , Z. , Ketchum , K.A. , Lai , Z. , Lei , Y. , Li , Z. , Li , J. , Liang , Y. , Lin , X. , Lu , F. , Merkulov , G.V. , Milshina , N. , Moore , H.M. , Naik , A.K. , Narayan , V.A. , Neelam , B. , Nusskern , D. , Rusch , D.B. , Salzberg , S. , Shao , W. , Shue , B. , Sun , J. , Wang , Z. , Wang , A. , Wang , X. , Wang , J. , Wei , M. , Wides , R. , Xiao , C. , Yan , C. , Yao , A. , Ye , J. , Zhan , M. , Zhang , W. , Zhang , H. , Zhao , Q. , Zheng , L. , Zhong , F. , Zhong , W. , Zhu , S. , Zhao , S. , Gilbert , D. , Baumhueter , S. , Spier , G. , Carter , C. , Cravchik , A. , Woodage , T. , Ali , F. , An , H. , Awe , A. , Baldwin , D. , Baden , H. , Barnstead , M. , Barrow , I. , Beeson , K. , Busam , D. , Carver , A. , Center , A. , Cheng , M.L. , Curry , L. , Danaher , S. , Davenport , L. , Desilets , R. , Dietz , S. , Dodson , K. , Doup , L. , Ferriera , S. , Garg , N. , Gluecksmann , A. , Hart , B. , Haynes , J. , Haynes , C. , Heiner , C. , Hladun , S. , Hostin , D. , Houck , J. , Howland , T. , Ibegwam , C. , Johnson , J. , Kalush , F. , Kline , L. , Koduru , S. , Love , A. , Mann , F. , May , D. , McCawley , S. , McIntosh , T. , McMullen , I. , Moy , M. , Moy , L. , Murphy , B. , Nelson , K. , Pfannkoch , C. , Pratts , E. , Puri , V. , Qureshi , H. , Reardon , M. , Rodriguez , R. , Rogers , Y.H. , Romblad , D. , Ruhfel , B. , Scott , R. , Sitter , C. , Smallwood , M. , Stewart , E. , Strong , R. , Suh , E. , Thomas , R. , Tint , N.N. , Tse , S. , Vech , C. , Wang , G. , Wetter , J. , Williams , S. , Williams , M. , Windsor , S. , Winn-Deen , E. , Wolfe , K. , Zaveri , J. , Zaveri , K. , Abril , J.F. , Guigo , R. , Campbell , M.J. , Sjolander , K.V. , Karlak , B. , Kejariwal , A. , Mi , H. , Lazareva , B. , Hatton , T. , Narechania , A. , Diemer , K. , Muruganujan , A. , Guo , N. , Sato , S. , Bafna , V. , Istrail , S. , Lippert , R. , Schwartz , R. , Walenz , B. , Yooseph , S. , Allen , D. , Basu , A. , Baxendale , J. , Blick , L. , Caminha , M. , Carnes-Stine , J. , Caulk , P. , Chiang , Y.H. , Coyne , M. , Dahlke , C. , Mays , A. , Dombroski , M. , Donnelly , M. , Ely , D. , Esparham , S. , Fosler , C. , Gire , H. , Glanowski , S. , Glasser , K. , Glodek , A. , Gorokhov , M. , Graham , K. , Gropman , B. , Harris , M. , Heil , J. , Henderson , S. , Hoover , J. , Jennings , D. , Jordan , C. , Jordan , J. , Kasha , J. , Kagan , L. , Kraft , C. , Levitsky , A. , Lewis , M. , Liu , X. , Lopez , J. , Ma , D. , Majoros , W. , McDaniel , J. , Murphy , S. , Newman , M. , Nguyen , T. , Nguyen , N. , and Nodell , M. ( 2001 ) . The sequence of the human genome . Science 291 , 1304 – 1351 . Weir , H.K. , Thun , M.J. , Hankey , B.F. , Ries , L.A. , Howe , H.L. , Wingo , P.A. , Jemal , A. , Ward , E. , Anderson , R.N. , and Edwards , B.K. ( 2003 ) . Annual report to the nation on the status of cancer, 1975 – 2000, fea- turing the uses of surveillance data for cancer prevention and control . J. Natl. Cancer Inst. 95 , 1276 – 1299 . Wenz , H.M. , Ramachandra , S. , O’Connell , C.D. , and Atha , D.H. ( 1998 ) . Identification of known p53 point mutations by capillary electro- phoresis using unique mobility profiles in a blinded study . Mutat. Res. 382 , 121 – 132 . Williams , L.C. , Hedge , M.R. , Herrera , G. , Stapleton , P.M. , and Love , D.R. ( 1999 ) . Comparative semi-automated analysis of (CAG) repeats in the Huntington disease gene: use of internal standards . Mol. Cell. Probes 13 , 283 – 289 . Woolley , A.T. , Hadley , D. , Landre , P. , deMello , A.J. , Mathies , R.A. , and Northrup , M.A. ( 1996 ) . Functional integration of PCR amplifica- tion and capillary electrophoresis in a microfabricated DNA analysis device . Anal. Chem. 68 , 4081 – 4086 . Woolley , A.T. , Sensabaugh , G.F. , and Mathies , R.A. ( 1997 ) . High-speed DNA genotyping using microfabricated capillary array electrophore- sis chips . Anal. Chem. 69 , 2181 – 2186 . Wu , R. , Hu , L. , Wang , F. , Ye , M. , and Zou , H. ( 2008 ) . Recent develop- ment of monolithic stationary phases with emphasis on microscale chromatographic separation . J. Chromatogr. A 1184 , 369 – 392 . Zhang , J. , Voss , K.O. , Shaw , D.F. , Roos , K.P. , Lewis , D.F. , Yan , J. , Jiang , R. , Ren , H. , Hou , J.Y. , Fang , Y. , Puyang , X. , Ahmadzadeh , H. , and Dovichi , N.J. ( 1999 ) . A multiple-capillary electrophoresis system for small-scale DNA sequencing and analysis . Nucleic Acids Res. 27 , e36 . Zhang , J. , Burger , C. , and Chu , B. ( 2006 ) . Nanostructured polymer matrix for oligonucleotide separation . Electrophoresis 27 , 3391 – 3398 . Zhu , Y. , Spitz , M.R. , Lei , L. , Mills , G.B. , and Wu , X. ( 2001 ) . A single nucleotide polymorphism in the matrix metalloproteinase-1 promoter enhances lung cancer susceptibility . Cancer Res. 61 , 7825 – 7829 . Zielenski , J. , Aznarez , I. , Onay , T. , Tzounzouris , J. , Markiewicz , D. , and Tsui , L.C. ( 2002 ) . CFTR mutation detection by multiplex heterodu- plex (mHET) analysis on MDE gel . Methods Mol. Med. 70 , 3 – 19 . Molecular Diagnostics76 the polymerase chain reaction (PCR) are subjected to electrophoresis through a linearly increasing gradient of temperature (or concentration gradient of denaturing agents such as urea and formamide for DGGE). Nucleotide substi- tutions and other small changes in the DNA sequence are associated with additional bands following TGGE. The electrophoretic mobility of DNA fragments differs according to whether the fragment is completely double helical, if one or more melting domains has dissociated, or if complete dissociation to two single-stranded molecules has occurred. Each of these states can be visualized using a perpendicular TGGE experiment, as will be discussed fur- ther below (see section 6.3.2). The electrophoretic mobility of a double helical (non- denatured) DNA fragment is not significantly altered by single-nucleotide substitutions within it, but is primarily dependent on the length and perhaps the curvature of the fragment ( Haran et al. , 1994 ). Therefore, assuming that PCR products contain a mixture of two DNA fragments that differ at a single position, as would be the case for a heterozygous point mutation, both fragments will initially progress through the gel at the same speed. When the molecules reach that point in the gel where the temperature equals their T m , the molecules will expe- rience a decrease in mobility owing to a transition from a completely duplex (double helical) conformation to a par- tially denatured one. Dissociation of the first or first few melting domains generally results in a dramatic reduction in the mobility of the DNA fragment, because the fragment takes on a complex, branched conformation. Due to the strong sequence dependence of the melt- ing temperature, branching (dissociation) and consequent retardation of electrophoretic mobility occurs at different levels of the temperature gradient associated with bands at different positions in the gel ( Myers et al. , 1987 ). In addi- tion to the two homoduplex molecules (wt/wt and mt/mt), two different heteroduplex molecules (wt/mt and mt/wt) can be formed by dissociating and reannealing DNA frag- ments containing a heterozygous mutation prior to per- forming TGGE ( Fig. 6.1 ). In practice, it is also possible to perform 40 cycles of PCR; the activity of the Taq polymer- ase is exhausted in the final cycles of PCR, such that het- eroduplices are formed as efficiently as if one performed denaturation and reannealing following PCR . Heteroduplex fragments then contain unpaired bases or “ bulges ” in the otherwise double helical DNA, resulting in a significant reduction in the T m of the affected melting domain ( Ke and Wartell, 1995 ). The melting temperatures of the two heteroduplex molecules are generally different from one another, so that each heteroduplex is separately visible in the gel. A heterozygous point mutation will thus be visu- alized by the appearance of four bands: a band represent- ing the normal allele (homoduplex), a band representing the mutant homoduplex that will lie above or underneath the wild-type homoduplex band, depending on the effect of the mutation on the T m , and two heteroduplex bands that are always above the homoduplex bands ( Fig. 6.2 ; Myers et al. , 1987 ). Mutant and wild-type homoduplex bands are separated by 2 – 10 mm in a typical polyacrylamide gel, and the heteroduplex bands are often three or more cm above the homoduplex bands. 6.2.3 Mutations are only Detectable in the Lowest Melting Domain(s) In the discussion above, a significant issue is that mutations are detectable only in the melting domain(s) with the lowest melting temperature. If, however, a DNA molecule contains several melting domains with different melting temperatures, it is generally not possible to visualize mutations located else- where than in the melting domain with the lowest T m . Once the DNA fragments reach the temperature at which the first melting domain dissociates, the mobility of the fragment is greatly reduced so that it may not reach temperatures relevant for the higher T m domains under the conditions of the experi- ment. Also, dissociation of the highest T m domain results in complete dissociation of the DNA fragment into two sin- gle-stranded DNA molecules. Single-stranded DNA, like completely double helical DNA, does not demonstrate dif- ferences in electrophoretic mobility, owing to small sequence changes, and hence there is no possibility of distinguishing two sequences once complete dissociation has occurred. The consequence of these observations is that only mutations in the lowest T m domain can be reliably detected by TGGE or DGGE ( Myers et al. , 1987 ). 6.2.4 GC- and Psoralen Clamps Extend the Usefulness of TGGE Myers and coworkers (1985a) presented an extension of the original DGGE protocol that allowed mutations in every region of the DNA fragment under analysis to be detected. FIGURE 6.1 Mechanism of heteroduplex formation. In the case of het- erozygous point mutations in genetic disorders, PCR produces two alleles differing only at the position of the point mutation. A wild-type (AA) and a mutant (aa) molecule are present at an approximately 1:1 ratio. Denaturation followed by reannealing of these molecules produces both wild-type (AA) and mutant (aa) homoduplex molecules, as well as two heteroduplex molecules, consisting of a wild-type and a mutant strand (Aa and aAa). Chapter | 6 Temperature and Denaturing Gradient Gel Electrophoresis 77 These researchers attached a 135 bp, GC-rich sequence, known as “ GC-clamp ” , to the β -globin promoter region in which mutations were being sought. The β -globin promoter region was found to contain two melting domains; with- out the GC-clamp, only mutations in the domain with the lower T m could be visualized in the gel. Owing to its high GC content, the GC-clamp has a significantly higher melt- ing temperature than most naturally occurring sequences. The attachment of the GC-clamp was found to significantly alter the melting properties of the β -globin sequence and mutations in the entire β -globin sequence could be experi- mentally detected ( Myers et al. , 1985a ). By adding a 40 nt G  C-rich sequence to one of the two PCR primers, a GC-clamp can be conveniently added to any DNA frag- ment produced by PCR ( Sheffield et al. , 1989 ). It is also possible to use a universal GC-clamp that is incorporated into amplified DNA fragments during PCR, thereby avoid- ing the expense of synthesizing long primers ( Top, 1992 ). Psoralen -modified PCR primers are an alternative to GC-clamps. One of the two PCR primers is 5  modified by 5-( ω -hexyloxy)-psoralen. The 5  terminus of the primer should have two adenosine residues; if the natural sequence does not have AA, this sequence should be appended to the specific DNA sequence of the primer. Psoralens are bifunc- tional photoreagents that can form covalent bonds with pyrimidine bases (especially thymidine). If intercalated at 5  -TpT in double helical DNA (this will be the complemen- tary sequence of the 3  terminus of the other strand follow- ing PCR), psoralen forms a covalent bond with thymidine after photoinduction ( Costes et al. , 1993b ). Photoinduction can be performed by exposing the PCR products to a source of UV light (365) for 5 to 15 minutes, which can be con- veniently done in the original PCR tubes or 96-well plate. In general, psoralen clamping provides comparable results to GC clamping, except that cross-linking of the PCR fragments is only approximately 85% efficient, so that one observes single-stranded, denatured DNA fragments run- ning below the main bands in the TGGE. Psoralen clamping is sometimes preferred over GC-clamping because the PCR is often easier to optimize, and bipolar clamping is possible if necessary (see below). Psoralen modification of primers is available from many commercial oligonucleotide sources. 6.3 THE PRACTICE OF TEMPERATURE GRADIENT GEL ELECTROPHORESIS Detailed protocols for TGGE and DGGE are available else- where ( Kang et al. , 1995 ; Murdaugh and Lerman, 1996 ). In the following paragraphs, the most important issues con- cerning how to set up TGGE or DGGE assays successfully are discussed, including especially the issues related to primer design and optimization procedures. Several points that apply only to DGGE are discussed in section 6.4. 6.3.1 Primer Design for TGGE/DGGE One of the first and most widely used computer programs to design primers for TGGE was the Melt87 package by Lerman and Silverstein (1987) . An updated version of this program (Melt94) is available at http://web.mit.edu/osp/ www/melt.html . The Melt87 program calculates the T m for each bp in the DNA fragment, i.e. the temperature at which 50% of the individual molecules are double helical and 50% of the molecules are in a fully disordered, melted state. The results of such a calculation are termed “ melting map ” ( Fig. 6.3 ). One notices that DNA fragments are typi- cally divided into distinct melting domains of about 50 to 300 bp in length, in which all base pairs have nearly identi- cal T m . The melting map demonstrates the lowest melting domain in the DNA fragment; as mentioned above, only mutations in this region will be visible by TGGE analysis. A further useful program in the Melt87 package is SQHTX. This program calculates the expected displacement in the gradient for a single-nucleotide mismatch (as would be the case for a heteroduplex molecule with a single-nucleotide FIGURE 6.2 Parallel TGGE/DGGE. Mutation screening is generally performed with the temperature or denaturing gradient parallel to the direction of electrophoresis. In this example, results of electrophoresis from top to bottom for a hypothetical family segregating an autosomal recessive disorder are shown. Cases 3 and 4 are normal, carrying only the wild-type allele ( “ d ” ). Cases 2, 5, and 6 are heterozygous for a point mutation resulting in the appearance of an additional homoduplex band ( “ c ” ), as well as two additional heteroduplex bands ( “ a ” and “ b ” ). Case 1, which is homozygous for the mutation, shows just the mutant homodu- plex band ( “ c ” ). Molecular Diagnostics78 substitution) at every position in the fragment. This analysis provides the clearest indication of the position in the frag- ment, where mutations will be detectable by TGGE analy- sis ( Lerman and Silverstein, 1987 ). Figure 6.4 provides an example of a displacement map calculated with SQHTX. The Melt87 programs are DOS-based and difficult to use for those with little experience with DOS and menu- based programs. Melt87 has no graphic capabilities of its own, and users need to process its output with a graphics program of their choice. For this reason, several freely available and proprietary programs have become available, which are significantly easier to use ( Table 6.1 ). Users should load a DNA sequence encompassing the DNA fragment to be analyzed (e.g. an exon with flanking intron sequences) together with about 100 nucleotides “ extra ” sequence to either side of the fragment of interest. The above- mentioned programs can be used to find primers that result in a DNA fragment with melting properties adequate for TGGE or DGGE. In general, some amount of trial and error is needed to find optimal primers for any given sequence. Users need to decide both the position of the forward and reverse primers as well as whether the GC-clamp is to be placed on FIGURE 6.3 Melting map. This graphic represents a fragment from exon 14 of the NF1 gene and was produced using TGGE-Star. Each tick on the x-axis represents a base pair. The base pairs are numbered from 1 to 195. The y-axis shows the temperature where the probability for a bp to be melted has the value 0.95, 0.75, 0.5, 0.25, and 0.05, respectively. The 5  -terminus of the fragment corresponds to a GC-clamp. Additionally, one can distinguish two further melting domains: from the 5  -terminus to the 50th bp and from the 50th bp to the 3  -terminus. The difference between these two melting domains is small and the sensitivity of TGGE is not disturbed. If the difference between these two plateaus in the curve were higher, both regions would need to be tested in two different PCR-TGGE steps. Mutations were detected in both regions of this fragment: three aster- isks above the x-axis mark positions of mutations detected with this assay. FIGURE 6.4 Displacement maps calculating using the program SQHTX, and graphic created with TGGE-Star. In the case of a heterozygous muta- tion, two heteroduplex bands occur. Heteroduplices do not migrate as far as the wild-type fragments because they “ melt ” at lower temperatures. The distance of heteroduplex bands and wild-type bands depends on the electrophoretic duration (x-axis) and the base position (y-axis). A muta- tion can only be detected, when the displacement is higher than the reso- lution of the gel. The color codes indicate different electrophoretic times, and the width of each band of color indicates the expected displacement (in arbitrary units) in the gel for a point mutation at the corresponding position in the sequence. Note : The e-book for this title, including full- color images, is available for purchase at www.elsevierdirect.com. TABLE 6.1 Programs for the design of PCR primers for use in TGGE/DGGE. Name Comment URL Melt94 DOS-based http://web.mit.edu/osp/www/melt.html TGGE-Star DOS-based, freely available user-friendly wrapper for Melt87 ( Gille and Gille, 2002 ) http://www.charite.de/bioinf/tgge Poland Server-based implementation of Poland’s algorithm ( Steger, 1994 ) http://www.biophys.uni-duesseldorf. de/local/POLAND/poland.html MELTingeny A commercial, Java-based GUI program with flexible routines for designing DGGE/TGGE primers http://www.ingeny.com WinMelt, MacMelt Commercial GUI programs for melting profile analysis http://www.medprobe.com