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PCR Protocols Second Edition

Edited by John M. S. Bartlett Department of Surgery, Glasgow Royal Infirmary, Glasgow, UK and David Stirling Department of Hematology, Royal Infirmary of Edinburgh, Edinburgh, UK

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PCR protocols / edited by John M.S. Bartlett, David Stirling. --2nd ed. p. cm. -- (Methods in molecular biology ; v. 226) Includes bibliographical references and index. ISBN 0-89603-642-1 (hardcover) (alk. paper); ISBN 0-89603-627-8 (combbound); 1-59259-384-4 (e-ISBN) ISSN 1064-3745 1. Polymerase chain reaction—Laboratory manuals. I. Bartlett, John M. S. II. Stirling, David. III. Series. QP606.D46P3595 2003 572'.43--dc21 2003042324 Preface

Since the discovery/invention of the polymerase chain reaction (PCR) in the mid-1980s, use of this technology has become so widespread that few researchers will progress far in their careers without exposure to PCR in one form or another. The robustness and exquisite sensitivity of PCR has led to the development of a multitude of protocols that exploit different aspects of this methodology. PCR has thus been used to analyze nucleic acids from all known phyla, and even some species extinct for hundreds of years. Disciplines as diverse as ecology, forensic medicine, food production, and archaeology employ PCR analyses as key components of population moni- toring, genetic fingerprinting, quality assurance, and research. The place of PCR as a cornerstone of current and future scientific endeavor is beyond question. Such has been the expansion in PCR methodologies that a revision of the previous edition of PCR Protocols dealing with PCR was considered essential. In undertaking this revision we were conscious that although much has changed, the fundamentals of PCR remain unaltered. Indeed in some areas, methods developed 5 or even 10 years ago remain as effective and useful as more recent developments. How- ever, in other areas rapid progress has led to exciting new advances. We are also conscious of a “PCR generation gap,” whereby many of the early developments in PCR have passed into folklore. The usefulness of this second edition of PCR Protocols to all researchers will, we trust, be enhanced by the provision of an introductory section dealing both with the history of PCR and the legal and practical issues surrounding the development of PCR-based methods today. Such is the diversity of the PCR methodologies currently available that it would be impossible to provide an exhaustive PCR manual that covers all aspects of the technology. Many of the sections we have included can be, and in some cases have been, expanded into separate publications. Therefore in producing PCR Protocols we have sought to select those techniques that form a core manual for both the PCR novice and the adept. The methods we have selected represent worked examples from many fields that can be reproduced and adapted for use within the reader’s laboratory. We have sought to provide both a primer to allow the reader to gain basic experience of different PCR techniques, and in-depth insight into a variety of the more complex applications of PCR. Each section has been introduced by an overview chapter whose aim is to provide researchers with an understanding of the applications and limitations of the techniques that follow. As with each volume in the Methods in Molecular Biology series, clear instructions for the performance of the various protocols is supplemented by additional technical notes that provide valuable insights into the working of the technique in question. Though often brief, these notes provide essential details that allow for a successful outcome.

v vi Preface

We would like to express our gratitude to each of the contributors to PCR Protocols, 2nd edition, who have been patient over the period required to collate their contributions. Finally, thanks are owed to our wives, Dorothy and Muriel, for their patience during the long hours we have spent on this volume. John M. S. Bartlett David Stirling Contents

Preface ...... v Contributors ...... xiii

PART I. INTRODUCTION TO PCR 1 A Short History of the Polymerase Chain Reaction John M. S. Bartlett and David Stirling ...... 3 2 PCR Patent Issues Peter Carroll and David Casimir ...... 7 3 Equipping and Establishing a PCR Laboratory Susan McDonagh ...... 15 4 Quality Control in PCR David Stirling ...... 21

PART II. PREPARATION OF NUCLEIC ACID TEMPLATES 5 Extraction of Nucleic Acid Templates John M. S. Bartlett...... 27 6 Extraction of DNA from Whole Blood John M. S. Bartlett and Anne White...... 29 7 DNA Extraction from Tissue Helen Pearson and David Stirling...... 33 8 Extraction of DNA from Microdissected Archival Tissues James J. Going...... 35 9 RNA Extraction from Blood Helen Pearson ...... 43 10 RNA Extraction from Frozen Tissue John M. S. Bartlett...... 45 11 RNA Extraction from Tissue Sections Helen Pearson ...... 47 12 Dual DNA/RNA Extraction David Stirling and John M. S. Bartlett ...... 49 13 DNA Extraction from Fungi, Yeast, and Bacteria David Stirling ...... 53 vii viii Contents

14 Isolation of RNA Viruses from Biological Materials Susan McDonagh ...... 55 15 Extraction of Ancient DNA Wera M. Schmerer ...... 57 16 DNA Extraction from Plasma and Serum David Stirling ...... 63 17 Technical Notes for the Detection of Nucleic Acids John M. S. Bartlett...... 65 18 Technical Notes for the Recovery and Purification of PCR Products from Acrylamide Gels David Stirling ...... 77

PART III. BASIC PCR METHODS 19 PCR Primer Design David L. Hyndman and Masato Mitsuhashi ...... 81 20 Optimization of Polymerase Chain Reactions Haiying Grunenwald ...... 89 21 Subcycling PCR for Long-Distance Amplifications of Regions with High and Low Guanine–Cystine Content: Amplification of the Intron 22 Inversion of the FVIII Gene David Stirling ...... 101 22 Rapid Amplification of cDNA Ends Xin Wang and W. Scott Young III...... 105 23 Randomly Amplified Polymorphic DNA Fingerprinting: The Basics Ranil S. Dassanayake and Lakshman P. Samaranayake ...... 117 24 Microsphere-Based Single Nucleotide Polymorphism Genotyping Marie A. Iannone, J. David Taylor, Jingwen Chen, May-Sung Li, Fei Ye, and Michael P. Weiner ...... 123 25 Ligase Chain Reaction William H. Benjamin, Jr., Kim R. Smith, and Ken B. Waites ...... 135 26 Nested RT-PCR in a Single Closed Tube Antonio Olmos, Olga Esteban, Edson Bertolini, and Mariano Cambra ...... 151 27 Direct PCR from Serum: Application to Viral Genome Detection Kenji Abe ...... 161 Contents ix

28 Long PCR Amplification of Large Fragments of Viral Genomes: A Technical Overview Raymond Tellier, Jens Bukh, Suzanne U. Emerson, and Robert H. Purcell...... 167 29 Long PCR Methodology Raymond Tellier, Jens Bukh, Suzanne U. Emerson, and Robert H. Purcell...... 173

PART IV. ULTRASENSITIVE AND QUANTITATIVE PCR 30 Qualitative and Quantitative PCR: A Technical Overview David Stirling ...... 181 31 Ultrasensitive PCR Detection of Tumor Cells in Myeloma Friedrich W. Cremer and Marion Moos ...... 185 32 Ultrasensitive Quantitative PCR to Detect RNA Viruses Susan McDonagh ...... 197 33 Quantitative PCR for cAMP RI Alpha mRNA: Use of Site-Directed Mutation and PCR Mimics John M. S. Bartlett...... 205 34 Quantitation of Multiple RNA Species Ron Kerr...... 211

PART V. TRANSCRIPTOME ANALYSIS 35 Differential Display: A Technical Overview John M. S. Bartlett...... 217 36 AU-Differential Display, Reproducibility of a Differential mRNA Display Targeted to AU Motifs Orlando Dominguez, Lidia Sabater, Yaqoub Ashhab, Eva Belloso, and Ricardo Pujol-Borrell ...... 225 37 PCR Fluorescence Differential Display Kostya Khalturin, Sergej Kuznetsov, and Thomas C. G. Bosch ...... 237 38 Microarray Analysis Using RNA Arbitrarily Primed PCR Steven Ringquist, Gaelle Rondeau, Rosa-Ana Risques, Takuya Higashiyama, Yi-Peng Wang, Steffen Porwollik, David Boyle, Michael McClelland, and John Welsh ...... 245 39 Oligonucleotide Arrays for Genotyping: Enzymatic Methods for Typing Single Nucleotide Polymorphisms and Short Tandem Repeats Stephen Case-Green, Clare Pritchard, and Edwin Southern ...... 255 x Contents

40 Serial Analysis of Gene Expression Karin A. Oien ...... 271

PART VI. MUTATIONS AND POLYMORPHISMS 41 Mutation and Polymorphism Detection: A Technical Overview Joanne Edwards and John M. S. Bartlett ...... 287 42 Combining Multiplex and Touchdown PCR for Microsatellite Analysis Kanokporn Rithidech and John J. Dunn ...... 295 43 Detection of Microsatellite Instability and Loss of Heterozygosity Using DNA Extracted from Formalin-Fixed Paraffin-Embedded Tumor Material by Fluorescence-Based Multiplex Microsatellite PCR Joanne Edwards and John M. S. Bartlett ...... 301 44 Reaction of Shadow Band Synthesis During PCR Amplification of Repetitive Sequences from Modern and Ancient DNA Wera M. Schmerer ...... 309 45 Degenerate Oligonucleotide-Primed PCR Michaela Aubele and Jan Smida...... 315 46 Mutation Detection Using RT-PCR-RFLP Hitoshi Nakashima, Mitsuteru Akahoshi, and Yosuke Tanaka ...... 319 47 Multiplex Amplification Refractory Mutation System for the Detection of Prothrombotic Polymorphisms David Stirling ...... 323 48 PCR-SSCP Analysis of Polymorphism: A Simple and Sensitive Method for Detecting Differences Between Short Segments of DNA Mei Han and Mary Ann Robinson ...... 327

PART VII. PCR-BASED SEQUENCING 49 Sequencing: A Technical Overview David Stirling ...... 337 50 Preparation and Direct Automated Cycle Sequencing of PCR Products Susan E. Daniels ...... 341 51 Nonradioactive PCR Sequencing Using Digoxigenin Siegfried Kösel, Christoph B. Lücking, Rupert Egensperger, and Manuel B. Graeber ...... 347 Contents xi

52 Direct Sequencing by Thermal Asymmetric PCR Georges-Raoul Mazars and Charles Theillet ...... 355 53 Analysis of Nucleotide Sequence Variations by Solid-Phase Minisequencing Anu Suomalainen and Ann-Christine Syvänen...... 361 54 Direct Sequencing with Highly Degenerate and Inosine-Containing Primers Zhiyuan Shen, Jingmei Liu, Robert L. Wells, and Mortimer M. Elkind ...... 367 55 Determination of Unknown Genomic Sequences Without Cloning Jean-Pierre Quivy and Peter B. Becker ...... 373 56 Cloning PCR Products for Sequencing in M13 Vectors David Walsh ...... 385 57 DNA Rescue by the Vectorette Method Marcia A. McAleer, Alison J. Coffey, and Ian Dunham ...... 393 58 Technical Notes for Sequencing Difficult Templates David Stirling ...... 401

PART VIII. IN SITU PCR AND PRINS 59 PCR-Based Detection of Nucleic Acids in Chromosomes, Cells, and Tissues: Technical Considerations on PRINS and In Situ PCR, and Comparison with In Situ Hybridization Ernst J. M. Speel, Frans C. S. Ramaekers, and Anton H. N. Hopman ...... 405 60 Cycling Primed In Situ Amplification John H. Bull and Lynn Paskins ...... 425 61 Direct and Indirect In Situ PCR Klaus Hermann Wiedorn and Torsten Goldmann ...... 433 62 Reverse Transcriptase In Situ PCR: New Methods in Cellular Interrogation Mark Gilchrist and A. Dean Befus ...... 445 63 Primed In Situ Nucleic Acid Labeling Combined with Immunocytochemistry to Simultaneously Localize DNA and Proteins in Cells and Chromosomes Ernst J. M. Speel, Frans C. S. Ramaekers, and Anton H. N. Hopman ...... 453 xii Contents

PART IX. CLONING AND MUTAGENESIS 64 Cloning and Mutagenesis: A Technical Overview Helen Pearson and David Stirling...... 467 65 Using T4 DNA Polymerase to Generate Clonable PCR Products Kai Wang ...... 469 66 A T-Linker Strategy for Modification and Directional Cloning of PCR Products Robert M. Horton, Raghavanpillai Raju, and Bianca M. Conti-Fine ...... 475 67 Cloning Gene Family Members Using PCR with Degenerate Oligonucleotide Primers Gregory M. Preston ...... 485 68 cDNA Libraries from a Low Amount of Cells Philippe Ravassard, Christine Icard-Liepkalns, Jacques Mallet, and Jean Baptiste Dumas Milne Edwards ...... 499 69 Creation of Chimeric Junctions, Deletions, and Insertions by PCR Genevieve Pont-Kingdon ...... 511 70 Recombination and Site-Directed Mutagenesis Using Recombination PCR Douglas H. Jones and Stanley C. Winistorfer ...... 517 71 Megaprimer PCR: Application in Mutagenesis and Gene Fusion Emily Burke and Sailen Barik ...... 525 Index ...... 533 Contributors

KENJI ABE • Department of Pathology, National Institute of Infectious Diseases, Tokyo, Japan MITSUTERU AKAHOSHI • Genetic Polymorphism and Function Assay Group, Genetic Polymorphism Institute, RIKEN Yokohama Institute, Yokohama, Japan YAQOUB ASHHAB • Unidad de Inmunología, Hospital Universitario Germans Trias i Pujol, and Faculty of Medicine, Universitat Autònoma de Barcelona, Barcelona, Spain MICHAELA AUBELE • Institute of Pathology, GSF-National Research Center for Environment and Health, Neuherberg, Germany SAILEN BARIK • Department of Biochemistry and Molecular Biology and Department of Microbiology and Immunology, University of South Alabama College of Medicine, Mobile, AL JOHN M. S. BARTLETT • Endocrine Cancer Group, Division of Cancer and Molecular Pathology, University Department of Surgery, Glasgow Royal Infirmary, Glasgow, United Kingdom PETER B. BECKER • Adolf-Butenandt-Institut, Ludwig Maximilians Universität, Munchen, Germany DEAN BEFUS • Glaxo-Heritage Asthma Research Laboratory, Pulmonary Research Group, Division of Pulmonary Medicine, Department of Medicine, University of Alberta, Edmonton, Alberta, Canada EVA BELLOSO • Unidad de Inmunología, Hospital Universitario Germans Trias i Pujol, and Faculty of Medicine, Universitat Autònoma de Barcelona, Barcelona, Spain WILLIAM H. BENJAMIN, JR. • Departments of Pathology and Microbiology, University of Alabama at Birmingham, Birmingham, AL EDSON BERTOLINI • Department of Plant Protection and Biotechnology, Instituto Valenciano de Investigaciones Agrarias, Valencia, Spain THOMAS C. G. BOSCH • Zoological Institute, University of Kiel, Kiel Germany DAVID BOYLE • Sidney Kimmel Cancer Center, San Diego, CA JENS BUKH • Hepatitis Viruses and Molecular Hepatitis Sections, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD JOHN H. BULL • CMC International Ltd, Macclesfield, Cheshire, UK EMILY BURKE • The Scripps Research Institute, La Jolla, CA MARIANO CAMBRA • Department of Plant Protection and Biotechnology, Instituto Valenciano de Investigaciones Agrarias, Valencia, Spain PETER CARROLL • Medlen & Carroll, LLP, Cambridge, MA STEPHEN CASE-GREEN • Department of Biochemistry, University of Oxford, Oxford, UK DAVID CASIMIR • Medlen & Carroll, LLP, Madison, WI xiii xiv Contributors

JINGWEN CHEN • Department of Technology Development, GlaxoSmithKline, Research Triangle Park, NC ALISON J. COFFEY • The Wellcome Trust Sanger Institute, Cambridge, UK BIANCA CONTI-FINE • Department of Biochemistry, University of Minnesota, St. Paul, MN FRIEDRICH W. CREMER • Department of Internal Medicine, University of Heidelberg, Heidelberg, Germany RANIL S. DASSANAYAKE • Faculty of Medicine, Department of Biochemistry and Molecular Biology, University of Colombo, Sri Lanka ORLANDO DOMINGUEZ • Programa de Biotechnologia, Centro Nacional de Investigaciones Oncologicas, Madrid, Spain JEAN BAPTISTE DUMAS MILNE EDWARDS • GENSET, Paris, France IAN DUNHAM • The Wellcome Trust Sanger Institute, Cambridge, UK JOHN J. DUNN • Biology Department, Brookhaven National Laboratory, Upton, NY JOANNE EDWARDS • Endocrine Cancer Group, Division of Cancer and Molecular Pathology, University Department of Surgery, Glasgow Royal Infirmary, Glasgow, United Kingdom RUPERT EGENSPERGER • Institute of Neuropathology, University of Münster, Münster, Germany MORTIMER M. ELKIND • Department of Radiological Health Sciences, Colorado State University, Fort Collins, CO SUZANNE U. EMERSON • Hepatitis Viruses and Molecular Hepatitis Sections, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD OLGA ESTEBAN • Department of Plant Protection and Biotechnology, Instituto Valenciano de Investigaciones Agrarias, Valencia, Spain MARK GILCHRIST • Glaxo-Heritage Asthma Research Laboratory, Pulmonary Research Group, Division of Pulmonary Medicine, Department of Medicine, University of Alberta, Edmonton, Alberta, Canada JAMES J. GOING • Department of Pathology, Glasgow Royal Infirmary, Glasgow, UK TORSTEN GOLDMANN • Clinical and Experimental Pathology, Research Center Borstel, Borstel, Germany MANUEL B. GRAEBER • Department of Neuropathology, Imperial College, London, UK HAIYING GRUNENWALD • Epicentre Technologies Corp., Madison, WI MEI HAN • Transplantation Immunology, Department of Internal Medicine, UT Southwestern Medical Center, Dallas, TX TAKUYA HIGASHIYAMA • Molecular Biology Program, Sidney Kimmel Cancer Center, San Diego, CA ANTON H. N. HOPMAN • Department of Molecular Cell Biology, Research Institute Growth & Development, University of Maastricht, The Netherlands ROBERT M. HORTON • Attotron Biosensor Corp., Carson City, NV DAVID L. HYNDMAN • Software Development, Automation Biology Drug Discovery Department, Ribapharm Inc., Costa Mesa, CA MARIE A. IANNONE • Department of Gene Expression and Protein Biochemistry, GlaxoSmithKline, Research Triangle Park, NC CHRISTINE ICARD-LIEPKALNS • GENSET, Paris, France Contributors xv

DOUGLAS H. JONES • Cedar Centre, Cedar Rapids, IA RON KERR • Department of Haematology, Royal Infirmary of Edinburgh, Edinburgh, UK KOSTYA KHALTURIN • Zoological Institute, University of Kiel, Kiel, Germany SIEGFRIED KÖSEL • Labor Becker, Olgemöller & Kollegen, Munich, Germany SERGEJ KUZNETSOV • Zoological Institute, University of Kiel, Kiel, Germany MAY-SUNG LI • Bio-Informatics Group Inc., NC JINGMEI LIU • Life Sciences Division, Los Alamos National Laboratory, Los Alamos, NM CHRISTOPH B. LÜCKING • Department of Neurology, University of Munich, Munich, Germany JACQUES MALLET • CNRS, Paris, France GEORGES-RAOUL MAZARS • Université Paul Sabatier, Tailaise, France MARCIA A. MCALEER • Department of Molecular Biology, Yamanouchi Research Institute, Oxford, UK MICHAEL MCCLELLAND • Molecular Biology Program, Sidney Kimmel Cancer Center, San Diego, CA SUSAN MCDONAGH • Department of Stomatology, University of California, San Francisco, CA MASATO MITSUHASHI • Hitachi Chemical Research Center, Irvine, CA MARION MOOSE • Department of Internal Medicine, University of Heidelberg, Heidelberg, Germany HITOSHI NAKASHIMA • Medicine and Biosystemic Science, Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan KARIN A. OIEN • CRC Department of Medical Oncology, University of Glasgow, Glasgow, UK ANTONIO OLMOS • Department of Plant Protection and Biotechnology, Instituto Valenciano de Investigaciones Agrarias, Valencia, Spain LYNN PASKINS • a-1 biotech UK, Bedfordshire, UK HELEN PEARSON • Molecular Endocrinology Unit, Western General Hospital, Edinburgh, UK GENEVIEVE PONT-KINGDON • ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT STEFFEN PORWOLLIK • Molecular Biology Program, Sidney Kimmel Cancer Center, San Diego, CA GREGORY M. PRESTON • Genomic and Proteomic Sciences, Pfizer Inc., Groton, CT CLARE PRITCHARD • Harwell Microarray Facility, MRC Mammalian Genetics Unit, Harwell, Oxfordshire, UK RICARDO PUJOL-BORRELL • Unidad de Inmunología, Hospital Universitario Germans Trias i Pujol, and Faculty of Medicine, Universitat Autònoma de Barcelona, Barcelona, Spain ROBERT H. PURCELL • Hepatitis Viruses and Molecular Hepatitis Sections, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD JEAN-PIERRE QUIVY • Institut Curie, Section de Recherche UMR 218, Paris France RAGHAVANPILLAI RAJU • Department of Biochemistry, University of Minnesota, St. Paul, MN xvi Contributors

FRANS C. S. RAMAEKERS • Department of Molecular Cell Biology, Research Institute Growth & Development, University of Maastricht, The Netherlands PHILIPPE RAVASSARD • GENSET, Paris, France STEVEN RINGQUIST • Sidney Kimmel Cancer Center, San Diego, CA ROSA-ANA RISQUES • Sidney Kimmel Cancer Center, San Diego, CA KANOKPORN RITHIDECH • Department of Pathology, State University of New York at Stony Brook, Stony Brook, NY MARY ANN ROBINSON • Research Technologies Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD GAELLE RONDEAU • Sidney Kimmel Cancer Center, San Diego, CA LIDIA SABATER • Unidad de Inmunología, Hospital Universitario Germans Trias i Pujol, and Faculty of Medicine, Universitat Autònoma de Barcelona, Barcelona, Spain LAKSHMAN P. S AMARANAYAKE • Oral Bio-Sciences, Faculty of Dentistry, The University of Hong Kong, Hong Kong, China WERA M. SCHMERER • Paleo DNA Laboratory, Department of Anthropology, Lakehead University, Thunder Bay, Ontario, Canada ZHIYUAN SHEN • Life Sciences Division, Los Alamos National Laboratory, Los Alamos, NM JAN SMIDA • Technische Universität München, Institute of Pathology, Munich, Germany KIM R. SMITH • Division of Infectious Diseases, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL EDWIN SOUTHERN • Department of Biochemistry, University of Oxford, Oxford, UK ERNST J. M. SPEEL • Research Institute Growth & Development, Department of Molecular Cell Biology, University of Maastricht, The Netherlands DAVID STIRLING • Department of Hematology, Royal Infirmary of Edinburgh, Edinburgh, UK ANU SUOMALAINEN • Programme of Neurosciences and Department of Neurology, Biomedicum-Helsinki, Helsinki University, Helsinki, Finland ANN-CHRISTINE SYVÄNEN • Department of Medical Sciences, Uppsala University Hospital, Uppsala, Sweden YOSUKE TANAKA • Department of Internal Medicine, Sawara Hospital, Meinohama Nishi-ku, Japan J. DAVID TAYLOR • Department of Technology Development, GlaxoSmithKline, Research Triangle Park, NC RAYMOND TELLIER • Division of Microbiology, Department of Paediatric Laboratory Medicine, The Hospital for Sick Children, Toronto, Ontario, Canada CHARLES THEILLET • INSERM, Cancer Center of Montpellier, Montpellier, France KEN B. WAITES, Departments of Pathology and Microbiology, University of Alabama at Birmingham, Birmingham, AL KAI WANG • PhenoGenomics Corp., Bothell, WA XIN WANG • CELERA, an Applera Corporation Business, Rockville, MD YI-PENG WANG • Molecular Biology Program, Sidney Kimmel Cancer Center, San Diego, CA Contributors xvii

DAVID WALSH • Crop Science Department, Scottish Agricultural College, Edinburgh, UK MICHAEL P. W EINER • 454 Corp., Branford, CT ROBERT L. WELLS • Radiological Health Sciences Department, Colorado State University, Fort Collins, CO JOHN WELSH • Molecular Biology Program, Sidney Kimmel Cancer Center, San Diego, CA ANNE WHITE • DNASHEF Technologies, Royal Infirmary of Edinburgh, Edinburgh, UK KLAUS HERMANN WIEDORN • Institut für Pathologie, Katharinenhospital Stuttgart, Stuttgart, Germany STANLEY C. WINISTORFER • Department of Physiology, University of Iowa, Iowa City, IA FEI YE • IcAgen Inc., Durham, NC W. SCOTT YOUNG III • Laboratory of Cellular and Molecular Regulation, National Institute of Mental Health, National Institutes of Health, Bethesda, MD (ISTORYOF0#2

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Index

A gel electrophoresis and analysis, Agarose gel electrophoresis, 324, 325 agarose concentration by molecular materials, 323, 324 weight range, 66 principles, 47 buffers, 66 Ancient DNA extraction, see DNA casting, 70, 74 extraction comparison with polyacrylamide gel Aquaporins, degenerate oligonucleotide- electrophoresis, 65, 66 primed PCR for gene family DNA recovery, 67, 71, 72, 74 member cloning, 486, 487, 493 ethidium bromide decontamination, ARMS, see Amplification refractory 71, 74 mutations system loading, 71, 74 Artifacts, PCR mutational analysis, 291 loading dyes, 67 ASO PCR, see Allele-specific materials, 69, 73 oligonucleotide PCR, running conditions, 71, 74 AU differential display, see Differential staining, 67, 71, 74 display Allele-specific oligonucleotide (ASO) B PCR, myeloma cell detection, Blood DNA extraction, see DNA complementary-determining region extraction identification, DNA sequencing, 189, 194 C product cloning and bacterial lysis, cDNA, see Complementary DNA 188, 189 Cloning PCR products, consensus RT-PCR, 188, 194 degenerate oligonucleotide-primed DNA extraction, 187, 188 PCR for gene family member materials, 186, 187 cloning, primer design, 186 advantages, 485, 486 principles, 185, 186 amplification reaction, 491, 495, quantitative PCR, 496 limiting dilutions, 191, 192, 194, 195 aquaporin gene family members, optimization of PCR, 194 486, 487, 493 primer design, 190, 191, 194 cloning of products, primer sensitivity testing, 192–194 insert preparation, 494 primer testing, 191, 195 ligation, 494, 495 Amplification refractory mutations plasmid miniprep for system (ARMS), sequencing, 495 multiplex detection of prothrombotic transformation of bacteria, 494, polymorphisms, 495 amplification reactions, 324 vector preparation, 494 533 534 Index

DNA isolation, 491 D gel electrophoresis and analysis, Degenerate oligonucleotide-primed PCR 491, 492, 496 (DOP-PCR), materials, 487–489, 495, 496 amplification reaction, 316, 317 overview, 485 applications, 315 primer design, 486, 487, 489–491 cloning of gene family members, secondary amplifications and advantages, 485, 486 product purification, 492, amplification reaction, 491, 495, 495, 496 496 M13, see DNA sequencing aquaporin gene family members, restriction site primers, 467 486, 487, 493 T4 DNA polymerase for clonable cloning of products, product generation, insert preparation, 494 amplification reaction, 470–472 ligation, 494, 495 end repair and blunt-end cloning, plasmid miniprep for 471–473 sequencing, 495 end repair and sticky-end cloning, 471, 473 transformation of bacteria, 494, materials, 470 495 overview, 467, 469 vector preparation, 494 primer design, 470 DNA isolation, 491 T-linker strategy, gel electrophoresis and analysis, amplification reactions, 478, 479, 482 491, 492, 496 digestion and cloning, 479–481 materials, 487–489, 495, 496 His-T-linkers, 475–478 overview, 485 ligation, 479, 482 primer design, 486, 487, 489–491 materials, 478 secondary amplifications and Nde-T-linker, 475, 476 product purification, 492, one-tube ligation and 495, 496 reamplification, 479, 482 gel electrophoresis, 316, 317 overview, 467, 475 materials, 315, 316 reamplification reaction, 479 topoisomerase treatment, 316, 317 Complementary DNA (cDNA), tumor sample preparation from library construction from low amount paraffin-embedded samples, 316 of cells, see SLIC strategy Differential display, rapid amplification of cDNA ends, AU-differential display, see Rapid amplification of rationale, 225 cDNA ends confirmation of differential Cyclic AMP RI alpha-binding proteins, expression, 232 see Quantitative reverse transcrip- criteria, 226 tion-polymerase chain reaction differential display, 228, 231, 234 Cytochrome P450, direct sequencing of gel electrophoresis, 228, 232, 235 PCR products using highly materials, 227, 228 degenerate and inosine-containing overview, 225–227 primers, 367–372 primers, 225, 226, 232, 233 Index 535

reverse transcription, 227, 228, section cutting, 36, 39 230, 234 section dewaxing and staining, 36, RNA preparation, 227–230, 233, 39 234 tungsten microneedle preparation, overview of transcriptome analysis, 36, 37, 40 219, 220 bacteria, 53, 54 PCR fluorescence differential bone marrow, 187, 188 display, dual DNA/RNA extraction, amplification reactions, 239, 240, diethylpyrocarbonate treatment, 49 242 DNA extraction, 50, 51 band elution and cloning, 240 materials, 49, 50 fluorescence imaging, 240 RNA extraction, 50, 51 gel electrophoresis, 240 fungi, 53, 54 materials, 237–240 mutation analysis purity criteria, 290, overview, 237, 238 291 reverse transcription, 239, 240 overview, 27 RNA isolation, 239, 240 plasma extraction, 63 primer design, 218, 219 serum extraction, 63 rationale, 217 tissue extraction, reverse transcriptases, 218 ethanol precipitation, 33, 34 tissue sources, 218 materials, 33 DNA chips, see Oligonucleotide arrays phenol/chloroform/isoamyl alcohol DNA extraction, extraction, 33 ancient DNA extraction from bone, proteinase K digestion, 33, 34 artifacts, 57 resuspension of DNA, 34 materials, 58 whole blood extraction, parameters based on DNA blood collection, 30 degradation state, 59 chloroform extraction, 30 phenol/chloroform/water ethanol precipitation, 30 extraction, 58–60 materials, 29, 30 powder preparation, 58 resuspension of DNA, 30, 31 precipitation, 58–60 yeast, 53, 54 safety, 60 DNA microarray, archived tissue samples, RNA arbitrarily primed PCR, see DNA purification, 38, 39 RNA arbitrarily primed PCR RNA analysis, 39 transcriptome analysis principles, frozen vs paraffin sections, 35, 39 220, 221 materials, 34 DNA polymerase, see T4 DNA microdissection, 37, 38, 40 polymerase; Taq polymerase microdissection tools, 36, 40 DNA rescue, see Yeast artificial proteinase K digestion, 38, 40 chromosome retrieval of dissected fragments, DNA sequencing, 38, 40 automation, 338, 339 RNA analysis, 39 chemical sequencing, 337, 338, 341 536 Index

criteria for PCR product sequencing, kinasing of primer, 380 339, 340 ligation, 380 cycle sequencing of PCR products, linker tag selection of PCR cycle sequencing, 343, 344 product, 381 isopropanol purification of DNA, materials, 376–378, 382, 383 342, 344 PCR, 380, 381, 383 materials, 342 primer purification and linker overview, 338, 341, 342 annealing, 379, 380 phenol/chloroform extraction of principles, 373–376 cycle sequencing products, template denaturation and 343, 344 sequencing reaction, 381–383 polyacrylamide gel M13 vector cloning of PCR products electrophoresis, 343, 344 for sequencing, detection of large inserts or deletions, ligation, 389, 390 289 M13 vector preparation, dideoxy sequencing, 338, 341, 361 dephosphorylation, 389 difficult templates, restriction digestion, 388, 390 A/T-rich sequences, 401 materials, 386, 387 high G/C content, 401 overview, 385, 386 homopolymer regions, 402 PCR products, secondary structure, 401 concatamerization and direct sequencing of PCR products, digestion, 388, 390 highly degenerate and inosine- purification, 387, 389 containing primer utilization, simultaneous end repair and labeling PCR, 370–372 materials, 369–371 phosphorylation, 387–389 principles, 367–369 nonradioactive PCR sequencing using template preparation, 370, 371 digoxigenin, overview, 339, 355, 356, 368 advantages, 347 thermal asymmetric PCR, controls, 347 annealing template and primer, materials, 347–349, 351, 353 358 polyacrylamide gel labeling reaction, 358 electrophoresis, materials, 356, 357 band visualization, 351, 353, PCR, 357, 358 casting, 350 sample preparation, 358 contact blotting, 351, 353 termination reactions, 358 running conditions, 350, 351 ligation-mediated PCR for unknown sequencing reactions, 350, 353 genomic sequences without template purification, 350, 352, cloning, 353 first primer extension, 380 solid-phase minisequencing, gel electrophoresis and analysis, affinity capture, 364, 365 382 applications, 361, 363 genomic DNA purification and calculations, 365, 366 restriction digestion, 379, 382 materials, 363–365 Index 537

PCR, 364 controls, 415, 418, 419 principles, 361, 362 direct PCR, DOP-PCR, see Degenerate amplification reactions, 438, 442 oligonucleotide-primed PCR contamination prevention, 436, E 441 controls, 439 Erlich, H., 4 deparaffinization, 437 F fixation, 436, 441 Flow cytometry, see Microsphere-based materials, 434–436, 440, 441 single nucleotide polymorphism nuclease treatment, 437, 438 genotyping overview, 415, 433 paraffin embedding, 436, 437 G permeabilization, 437, 441 Gel electrophoresis, see Agarose gel primer design, 440 electrophoresis; Polyacrylamide quenching of endogenous gel electrophoresis peroxidase, 438, 441 Gene fusion, see Megaprimer PCR reverse transcription, 438 H sample preparation, cytospins, 437 Hepatitis virus, tissue sections, 437, 440, 441 direct nested RT-PCR of serum washing and detection, 439, 442 samples for detection, indirect PCR, amplification reactions, 162, 164, amplification reactions, 438, 442 166 contamination prevention, 436, gel electrophoresis, 164 441 materials, 161–163 controls, 439 overview, 161, 165 deparaffinization, 437 pretreatment of samples, 162, 164 fixation, 436, 441 long reverse transcription-polymerase in situ hybridization, 438, 439, 442 chain reaction, 169 materials, 434–436, 440, 441 Historical perspective, PCR, 3–5 nuclease treatment, 437, 438 I overview, 415, 417, 433, 434 Immunocytochemistry, see Primed in paraffin embedding, 436, 437 situ labeling permeabilization, 437, 441 In situ hybridization (ISH), primer design, 440 controls, 415 quenching of endogenous indirect in situ PCR, 438, 439, 442 peroxidase, 438, 441 in situ PCR comparison, 411–414, reverse transcription, 438 420 sample preparation, primed in situ labeling comparison, cytospins, 437 406, 407, 412, 413, 419, 420 tissue sections, 437, 440, 441 sensitivity, 405, 406, 419 washing and detection, 439, 442 In situ PCR (IS-PCR), in situ hybridization comparison, applications, 440 411–414, 420 538 Index

primed in situ labeling comparison, applications, 135, 138–140 411–414, 420 clinical specimen preparation, 142 reverse transcription coupling, contamination control, 137–140, 146, amplification reactions, 449, 451 147 cell fixation, 447, 448, 450 detection of products, 137, 142, 143 controls, 449, 451 equipment, 139 digoxigenin detection and color gap ligase chain reaction, 136 development, 449 gel electrophoresis, 137 materials, 446, 447 inhibitors, 138 overview, 445, 446 kits for microbe detection, 138, 139, pretreatments, 448 146 prospects, 449, 450 ligase detection reaction, 136 protease digestion, 448, 450, 451 ligases, 136 reverse transcription, 448, 449, Neisseria gonorrheae detection, 451 Abbott LCx assay, sample fixation and processing, 414 amplification reactions, 145 sensitivity, 418 calculations, 146 Intellectual property, see Patents, PCR controls, 147 ISH, see In situ hybridization, detection of products, 145 Island rescue PCR, see Yeast artificial interpretation, 146 chromosome overview, 143 IS-PCR, see In situ PCR quality control, 144, 145, 147 K urine specimens, collection, 143 Khorana, G., 13 preparation and processing, 144 Kornberg, A., 10 storage, 143, 147 L swab specimens, 144 Laboratory set-up, PCR, primer selection, 136, 137, 140, 142 amplification lab, 18 principles, 135, 136 decontamination of equipment, 16, 18 reagents and supplies, 140 equipment, target sequence selection, 137 consumables, 17, 18 Ligation-mediated PCR, see DNA hoods, 16, 17 sequencing pipets, 16, 18 LOH, see Loss of heterozygosity table, 17 Long PCR, thermocyclers, 15, 16 amplification reactions, 174, 175 layout, 17 applications, 168, 173 processing area, 18 definition, 89 reagent preparatory lab, 17, 18 DNA polymerases, 167, 168, 173 sample preparation lab, 18 materials, 173–175 workflow, 15, 16 nested PCR, 174, 176 LCR, see Ligase chain reaction principles, 167, 168 Ligase chain reaction (LCR), Long reverse transcription-polymerase amplification reactions, 142 chain reaction, Index 539

amplification reactions, 174, 175 materials, 125, 126, 130 reverse transcription, 174, 176 microsphere coupling to RNA virus detection, 168, 169 cZipCodes, 127, 128 Loss of heterozygosity (LOH), target probe generation, 128 microsatellite analysis, 289–291, thermal cycling, 128 301, 302, 305–307 principles, 123–125 M single-base chain extension, capture probe hybridization to M13, see DNA sequencing microspheres, 129 Megaprimer PCR, flow cytometry and analysis, 129, materials, 527–529 130, 132 megaprimer synthesis, 527–529 materials, 127, 130 overview, 525 microsphere coupling to primers, 525, 527–529 cZipCodes, 127, 128 second amplification reaction, target probe generation, 128 mutant synthesis, 528–530 MT-PCR, see Multiplex-touchdown yield optimization, 525–527 PCR Microsatellite analysis, CAG repeats, 301 Mullis, K., 3, 4 fluorescence-based multiplex Multiple myeloma, see Allele-specific microsatellite PCR of paraffin- oligonucleotide PCR embedded tumor samples, Multiplex PCR, amplification reactions, 304, 305 definition, 89 gel electrophoresis, 305 multiplex microsatellite PCR, see loci in analysis, 304 Microsatellite analysis loss of heterozygosity analysis, primer design, 88 302, 305–307 Multiplex-touchdown PCR (MT-PCR), materials, 303, 304 advantages, 297 microdissection of archival DNA, hot start PCR, 296 304 master mix, 296 microsatellite instability analysis, materials, 295, 296 302 microsatellite analysis, 295 throughput, 302 overview, 295, 298 loss of heterozygosity, 289–291 polyacrylamide gel electrophoresis, multiplex-touchdown PCR, see 297 Multiplex-touchdown PCR touchdown cycles, 297 phylogeny studies, 292 rationale, 301 N Microsphere-based single nucleotide Neisseria gonorrheae detection, see polymorphism genotyping, Ligase chain reaction oligonucleotide ligation assay, Nested reverse transcription-polymerase capture probe hybridization to chain reaction, microspheres, 128 advantages, 156 flow cytometry and analysis, 128, amplification reaction, 156–158 129, 132 applications, 154 540 Index

direct RT-PCR of serum samples for Optimization, PCR parameters, hepatitis virus detection, allele-specific oligonucleotide PCR, amplification reactions, 162, 164, 194 166 buffers, 96 gel electrophoresis, 164 cosolvents, 95, 96 materials, 161–163 deoxynucleotide concentrations, 92 overview, 161, 165 hot start PCR, 96 pretreatment of samples, 162, 164 magnesium concentration, 92 gel electrophoresis, 156 materials, 90 immunocapture phase, 154, 156, 157 mixed template competition, 182 materials, 154, 156 overview, 89, 90 primer design, 155, 157 polymerase buffers, 92, 93 principles, 151–153 primer design, 91, 92 reaction mixes, 155, 156 reaction mixture, 90 reverse transcription, 156 shadow band prevention, see Shadow sensitivity, 159 bands single closed Eppendorf tube, 153, template quality and concentration, 156 91, 181 Nucleic acid extraction, see DNA thermal cycling, 90, 91, 93–95 extraction; RNA extraction troubleshooting, 96, 97 verification with gel electrophoresis, O 91 Oligonucleotide arrays, applications, 255, 288, 289 P biallelic single nucleotide PAGE, see Polyacrylamide gel polymorphism typing, 257, 258 electrophoresis enzyme-aided assays, Paraffin section nucleic acid extraction, ligation, 264, 266–268 see DNA extraction; RNA primer extension, 264, 266, 268 extraction short tandem repeat typing, 260, Patents, PCR, 262 abundance with PCR technology, 7–9 single nucleotide polymorphisms, academic researcher exemptions, 10, 260, 262 11 fabrication, 255–257, 263, 265 obtaining rights to practice PCR, 8, short tandem repeat arrays, 10 fabrication, 258, 265 validity, 11–14 hybridization assays, 258, 260, Plasma DNA extraction, see DNA 262, 264, 266, 267 extraction materials, 263, 264 Polyacrylamide gel electrophoresis target DNA preparation, 258, 263, (PAGE), 265, 266 acrylamide percentage by DNA size Oligonucleotide ligation assay, see range, 68 Microsphere-based single autoradiography, 68 nucleotide polymorphism comparison with agarose gel genotyping electrophoresis, 65–67 Index 541

denaturing gel electrophoresis, 68, overview, 410, 453, 454 70, 72–75 rationale, 453, 454 DNA recovery, 68, 70, 73, 74, 77, 78 in situ hybridization comparison, 406, fluorescent-labeled nucleotide 407, 412, 413, 419, 420 detection, 69 in situ PCR comparison, 411–414, materials, 69, 70, 73 420 nondenaturing gel electrophoresis, multicolor detection, 409 68–70, 72, 74 primers, 409 resolution, 67 principles, 425, 453 RNA arbitrarily primed PCR, 248 sample fixation and processing, 407, sequencing gels, see DNA 409 sequencing sensitivity improvement, 410, 411 Primed in situ labeling (PRINS), Primer design, PCR, applications, 425, 453 allele-specific oligonucleotide PCR, controls, 415 186, 190, 191, 194 cycling PRINS, amplicon size, 81 amplification reactions, 429, 430 AU-differential display, 225, 226, cell preparation, 428, 430 232, 233 detection, 429–431 cycling primed in situ labeling, 428 fixation, 428, 430 guanine/cytosine content, 81 materials, 427, 428, 430 hairpin avoidance, 84 microwave pretreatment, 429 hybridization simulation, 86 overview, 425, 427 primer design, 428 ligase chain reaction, 136, 137, 140, hybridization conditions, 409 142 immunocytochemistry combination, location, 81 fluorescence immunophenotyping melting temperature, 82, 83, 85 with alkaline phosphatase multiplex PCR, 88 Fast Red staining, nested reverse transcription- DNA labeling with PRINS, 458, polymerase chain reaction, 155, 459, 461, 462 157 materials, 454, 455, 455, 456 optimization, 91, 92 slide preparation, 458, 461 primer-dimer formation, 84, 85 metaphase chromosome protein recombination PCR, localization, chimeric primer, 512, 514 DNA labeling with PRINS, 460, 462 outside primers, 513, 514 immunofluorescence detection, software, 88 460, 461 solid-phase minisequencing, 364 materials, 457, 458 specificity, 85–88 mitotic spindle localization, 3'-terminal stability, 85, 87 DNA labeling with PRINS, 459, T4 DNA polymerase for clonable 461, 462 product generation, 470 immunofluorescence detection, three-step PCR, 83 459–461 two-step PCR, 83, 84 materials, 456, 457 PRINS, see Primed in situ labeling 542 Index

Q R Quality control, PCR, RACE, see Rapid amplification of contamination, 22 cDNA ends equipment, 21 Randomly amplified polymorphic DNA experimental controls, 22 (RAPD), ligase chain reaction, 144, 145, 147 Candida analysis, 119, 120 operator errors, 22 DNA polymerases, 118, 120 reagents, 21, 22 ionic composition, 120 regional quality-assurance programs, materials, 118, 119 22, 23 primer:template ratio, 118, 120 Quantitative PCR, primers, 117, 118 competitive template PCR, 183 principles, 117 end-point analysis, 182 template, 121 limiting dilution, 182 thermal cycling, 117–121 multiple myeloma cell detection, see RAPD, see Randomly amplified Allele-specific oligonucleotide polymorphic DNA PCR RAP-PCR, see RNA arbitrarily primed real-time PCR, 183 PCR Quantitative reverse transcription- Rapid amplification of cDNA ends polymerase chain reaction, (RACE), cyclic AMP RI alpha-binding protein expression profiling, 107, 113 transcripts, materials, 106, 107, 112, 113 amplification reactions, 206, 207, 209 principles, 105, 106 calculations, 208, 210 3'-RACE using tailed oligo-dT primer materials, 205, 206, 209 for cDNA synthesis, 111–114 overview, 205, 208, 209 5'-RACE using gene-specific primer restriction digestion, 207, 209, 210 for cDNA synthesis, 112–114 reverse transcription, 206, 209 universal oligo-dT primer for cDNA sensitivity, 208, 209 synthesis, 107–111, 113, 114 multiple RNA species quantification, Recombination PCR, see also Site- amplification reactions, 213 directed mutagenesis, dilutions, 213, 214 applications, 511, 514 internal controls, 211, 213, 214 chimeric junctions, deletions and materials, 211, 212 insertions generation, principles, 211 amplification reactions, 513, 514 reverse transcription, 213 materials, 511, 512 RNA virus detection, overview, 511 external standard curves, 198, 201 primer design, internal reference template, 198, chimeric primer, 512, 514 199, 201 outside primers, 513, 514 limiting dilution, 197, 198, 200, 201 principles, 517 materials, 199–201 Restriction fragment length overview, 197 polymorphism (RFLP), reverse transcription, 199–201 principles, 319 Index 543

reverse transcription-PCR coupling, overview, 27 gel electrophoresis and analysis, paraffin-embedded section extraction, 320–322 chloroform/isoamyl alcohol materials, 319, 320 extraction, 48 mismatch primers, 319 fixation effects on recovery, 47 restriction enzyme digestion, 320 materials, 47 reverse transcription, 320 precipitation, 48 Reverse transcription-polymerase chain SLIC strategy, 505 reaction (RT-PCR), see In situ viral RNA, PCR; Long reverse transcription- chloroform/isoamyl alcohol polymerase chain reaction; Nested extraction, 56 reverse transcription-polymerase ethanol precipitation, 56 chain reaction; Quantitative materials, 55 reverse transcription-polymerase phenol/chloroform extraction, 56 chain reaction; Restriction proteinase K treatment, 56 fragment length polymorphism; RT-PCR, see Reverse transcription- RNA arbitrarily primed PCR polymerase chain reaction RFLP, see Restriction fragment length polymorphism S RNA arbitrarily primed PCR (RAP-PCR), SAGE, see Serial analysis of gene amplification reactions, 248, 252 expression fluorescent labeling, 248, 250, 252 Scharf, F., 5 materials, 246, 247 Sequencing, see DNA sequencing microarray analysis advantages, 245 Serial analysis of gene expression microarray hybridization and data (SAGE), analysis, 250, 251 advantages and limitations, 221, 222 polyacrylamide gel electrophoresis, 248 bioinformatics, 275, 280 principles, 245 complementary DNA, reverse transcription, 248, 251, 252 binding to magnetic beads, 276 RNA preparation, 247, 248 ditag construction, 277 RNA extraction, ligation of sticky-end linkers, 276 blood samples, 43, 44 sticky-end generation, 276 dual DNA/RNA extraction, synthesis, 276, 281 diethylpyrocarbonate treatment, 49 tag creation and release from DNA extraction, 50, 51 magnetic beads, 276 materials, 49, 50 concatemers, RNA extraction, 50, 51 cloning, 279 frozen tissue extraction, DNA sequencing, 279, 280 diethylpyrocarbonate treatment, 45 gel purification, 278 disaggregation of tissue, 46 generation, 278 materials, 45 polymerase chain reaction phenol extraction, 46 screening of long concatemer precipitation, 46 inserts, 279 mutation analysis purity criteria, 290, differential display comparison, 220, 291 221 544 Index

ditags, materials, 330, 332 gel purification, 278 polyacrylamide gel electrophoresis polymerase chain reaction, 277, and autoradiography, 327– 278, 282 329, 331–333 restriction digestion and linker principles, 327, 330 removal, 278 screening for single nucleotide I-SAGE, 271, 273, 280 polymorphisms, 287, 288, 327 Internet resources, 273 Single strand ligation to single-stranded materials, 273–275, 281 DNA, see SLIC strategy principles, 221, 222, 271, 272 Site-directed mutagenesis, RNA preparation, 275, 276, 280, 281 overview of PCR approaches, 468, software, 271–273, 280 517 Serum, recombination PCR, direct RT-PCR for viral detection, amplification reaction, 519 amplification reactions, 162, 164, gel electrophoresis of products, 166 520 gel electrophoresis, 164 materials, 519, 522, 523 materials, 161–163 overview, 517–519 overview, 161, 165 plasmid digestion, 519, 523 pretreatment of samples, 162, 164 screening for mutations, 522 DNA extraction, see DNA extraction sequence verification, 522, 523 Shadow bands, transformation of bacteria, origins, 309 520–522 reduction strategy, 309–311 SLIC strategy, cDNA library Single-base chain extension, see construction, Microsphere-based single biotinylated oligonucleotide nucleotide polymorphism screening of library, 502, genotyping 507–509 Single nucleotide polymorphism blunt-end cloning of amplification genotyping, see Amplification products, 508 refractory mutations system; direct PCR on colonies, 508, 509 Ligase chain reaction; library amplification, 501, 502 Microsphere-based single materials, 503–505, 509 nucleotide polymorphism overview, 499 genotyping; Oligonucleotide PCR, 507, 509 arrays; Restriction fragment length primer removal, 506 polymorphism; Single-strand RNA extraction, 505 conformation polymorphism single-stranded cDNA ligation to Single-strand conformation modified oligonucleotide, 507, polymorphism (SSCP), 509 detection techniques, 287, 288, 327, single-stranded cDNA synthesis, 501, 328 505, 506, 509 polymerase chain reaction, size bias from PCR amplification, amplification, 330, 332 499 Index 545

subtractive library synthesis, 502 V Solid-phase minisequencing, see DNA Viral RNA, sequencing extraction, see RNA extraction S-PCR, see Subcycling PCR long reverse transcription-polymerase SSCP, see Single-strand conformation chain reaction, 168, 169 polymorphism quantitative reverse transcription- Subcycling PCR (S-PCR), polymerase chain reaction diagnostic applications, 101 detection, hemophilia A mutations, external standard curves, 198, 201 gel electrophoresis, 103 internal reference template, 198, materials, 101, 102 199, 201 reaction mixture, 102 limiting dilution, 197, 198, 200, thermal cycling, 102, 103 201 T materials, 199–201 T4 DNA polymerase, see Cloning PCR overview, 197 products reverse transcription, 199–201 Taq polymerase, Y fidelity, 93 history of use, 4 YAC, see Yeast artificial chromosome optimization, 93 Yeast artificial chromosome (YAC), patents, 8, 9, 12 contigs, 393 thermostability, 93 DNA rescue by vectorette method, Thermal asymmetric PCR, see DNA gel electrophoresis, 397 sequencing island rescue PCR, 394, 395 Thermocycler, materials, 395, 396 historical perspective, 5 PCR, 397, 398 laboratory set-up, 15, 16 principles, 393–395 optimization of cycling program, 93– restriction digestion, 396, 398 95 sample preparation, 396, 398 T-linker, see Cloning PCR products insert capacity, 393