THE NUCLEIC ACID PROTOCOLS HANDBOOK

TheNucleic Acid

ProtocolsHandbook

Edited by

Ralph RapleyUniversity ofHertfordshire

HatfieldUK

Humana Press *- Totowa, New Jersey

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The nucleic acid protocols handbook 1 edited by Ralph Rapley.p. em.

Includes index.ISBN 0-89603-459-3 (alk. paper) (hardcover), ISBN 0-89603-841-6 (paper)I. Nucleic acids~Laboratorymanuals. 2. Polymerase chain reaction~Laboratory manu­

als. 3. Gene mapping~Laboratorymanuals. 4. Molecular genetics~Laboratorymanuals.I. Rapley, Ralph.QP620.N7987 1999572.8---dc21 98-43385

CIP

Preface

There can be no doubt that some of the most spectacular advances made in scienceover the past few decades have been in the isolation, analysis, and manipulation ofnucleic acids. This has led to a much greater understanding ofmechanisms and processesacross many fields of bioscience, such as biochemistry, microbiology, physiology,pharmacology, and the medical sciences to name a few. It has also led to the growth ofthe biotechnology industry, which seeks to develop and commercialize many of theseimportant processes and methods. Much of this has come about because of the devel­opment of numerous molecular biology and genetic manipulation techniques. Thediscovery of restriction enzymes and the development of cloning vectors in the early1970s opened the door to ways ofisolating and manipulating nucleic acids that had neverbeen thought possible. Gene probe labeling and hybridization were developed andrefined to provide powerful methods ofanalysis. These-together with the developmentof DNA sequencing methods, protein engineering techniques, and PeR-have allcontinued to contribute substantially to the understanding ofbiological processes at themolecular level. The protocols for these important methods are the focus ofThe NucleicAcid Protocols Handbook, whose aim is to provide a comprehensive set of techniquesin one volume that will enable the isolation, analysis, and manipulation ofnucleic acidsto be readily undertaken.

The Nucleic Acid Protocols Handbook is divided into 10 parts; within each there areapproximately 10 chapters. The first four parts follow one another logically: nucleic acidextraction (Part I), basic separation and analysis ofDNA (II), through probe design andlabeling (III), and RNA analysis techniques (IV). The following three sections deal withgene library construction and screening (V), DNA sequencing (VI), and the polymerasechain reaction (VII). Part VIII deals with the analysis of genes, mutations, and proteininteractions and is followed by Part IX, on mutagenesis, transcription, and translation invitro. This is followed finally by Part X, on gene localization and mapping in situ.In compiling this volume a number of techniques have been drawn and updated fromversions appearing in earlier volumes of Humana Press' Methods in Molecular Biologyseries. These highly successful books have provided numerous laboratories with thetechniques needed to undertake modern laboratory molecular biology successfully. As such, their format has been followed in The Nucleic Acid Protocols Handbook.Thus a short introduction to the basic theory ofthe technique is followed by a completelisting of all materials and reagents needed before a particular protocol is presented.Step-by-step instructions are then provided in the Methods section. In addition, Notes arecited throughout the Methods and appear at the end of the chapter, providing valuableand highly useful information not found in traditional scientific literature. This essential

v

vi Preface

infonnation in many cases may mean the difference between the success or failure ofa particular technique and is one of the recognized key points of the Humana Methodsin Molecular Biology series.

It is inevitable that a degree ofoverlap occurs between some of the chapters. Indeed,the use ofthe polymerase chain reaction is now so widespread that it is a key elementof many of the protocols. These have been cross referenced where possible, althoughmost of the protocols are self-contained and can be attempted without the need to readfurther chapters. For those new or unfamiliar to laboratory molecular biology, thecompilation of protocols in The Nucleic Acid Protocols Handbook also provides theability to attempt protocols confidently. The intent was not to list all protocols in molec­ular biology (within one volume, this is an impossible task), and certainly moreadvanced protocols may be found in a number of excellent texts including many in theHumana Methods in Molecular Biology series. It was, however, the aim to provide themost commonly used protocols and alternatives in one volume at a level accessible tomost laboratories, which we believe has been achieved. In such a large compilation,much credit must go to the authors, who have devoted valuable time and effort to writeand update these protocols; to Prof. John M. Walker, the series editor, for his helpfuladvice and guidance; and to the staffat Humana Press for their substantial efforts in theproduction of the volume.

Ralph Rapley

Contents

Preface vContributors xv

PART I NUCLEIC ACID EXTRACTION

Isolation of High-Molecular-Weight DNA from Animal Cells 3Ian Garner

2 Isolation of mRNA by Affinity Chromatography 9Sian Bryant and David L. Manning

3 Isolation and Purification of DNA from Plants 13Justin Stacey and Peter G. Isaac

4 Purification of Uncontaminated, Intact Plant RNA 17Shu-Hua Cheng, Brandon D. Moore, and Jeffrey R. Seemann

5 An Improved Method to Isolate Mitochondrial RNAfrom Green Plant Tissue 23Fei Ye and Ral' Reski

6 Isolating Chromosomal DNA from Bacteria 29Elisabeth Chachaty and Patrick Saulnier

7 Bacterial DNA Extraction for Polymerase Chain Reactionand Pulsed-Field Gel Electrophoresis 33Elisabeth Chachaty and Patrick Saulnier

8 Isolation of Fungal Nucleic Acids 37Surapareddy Sreenivasaprasad

9 Total RNA Isolation from Bacteria 47John Heptinstall

10 Simultaneous RNA and DNA Extractionfrom Biopsy Material, Culture Cells, Plants, and Bacteria 53Udo Dobbeling

11 Spectrophotometric Analysis of Nucleic Acids 57John Heptinstall and Ralph Rapley

PART II BASIC SEPARATION AND ANALYSIS OF DNA

12 Restriction Endonuclease Digestion of DNA 63Duncan R. Smith

13 Agarose Gel Electrophoresis of Nucleic Acids 67D. Ross Williams and Ralph Rapley

vii

viii Contents

14 Preparation of RNA Dot Blots 71Rachel Hodge

15 Native Polyacrylamide Gel Electrophoresis 73Adrian J. Harwood

16 Southern Blotting of Agarose Gels by Capillary Transfer 77Ralph Rapley and Jane Davenport-Jones

17 Pulsed-Field Gel Electrophoresis 81John Maule

18 HPLC of DNA and PCR Products 105Elena D. Katz

PART III PROBE DESIGN, SYNTHESIS, AND LABELING

19 End-Labeling of DNA Fragments 117Adrian J. Harwood

20 Nick Translation and Random Hexamer Labeling of DNA 123Jane Davenport-Jones

21 Generation of Labeled Probes by Polymerase Chain Reaction 127Y. M. Dennis Lo and Shu F. An

22 Nonradioactive Oligonucleotide Probe Labeling 135Sue Fowler and Ian Durrant

23 Preparation of Direct, Enzyme-Labeled DNA Probes 145Ian Durrant and Timothy Stone

24 Random Prime Labeling of DNA Probeswith Fluorescein-Tagged Nucleotides 149Bronwen M. Harvey, Claire B. Wheeler, and Martin W. Cunningham

25 Hybridization and Detection of Fluorescein-Labeled DNA ProbesUsing Chemiluminescence 153Claire B. Wheeler, Bronwen M. Harvey, and Martin W. Cunningham

26 Hybridization of Enzyme-Labeled Probesand Detection by Chemiluminescence 157Timothy Stone and Ian Durrant

27 Hybridization and Competition Hybridization of Southern Blots 163Rosemary Kelsell

28 Autoradiography and Fluorography 169Eric Quemeneur

PART IV RNA ANALYSIS TECHNIQUES

29 Formaldehyde Gel Electrophoresis of Total RNA 177Sian Bryant and David L. Manning

30 RNA Probes for the Analysis of Gene Expression 181Dominique Belin

31 Primer Extension Analysis of mRNA 195Maggie Walmsley, Mark Leonard, and Roger Patient

Contents ix

32 S1 Mapping Using Single-Stranded DNA Probes 201Stephane Viville and Roberto Mantovani

33 Measurements of Rate of Transcription in Isolated Nucleiby Nuclear "Run-Off" Assay 207Rai Ajit K. Srivastava and Gustav Schonfeld

34 One-Tube RT-PCR with Sequence-Specific RT Primers 213Ulrich Pfeffer and Paola Ferro

35 Characterization of RNA Using Continuous RT-PCRCoupled with ELOSA 219Fram;ois Mallet

36 Quantitative Analysis of RNA Species by Polymerase ChainReaction and Solid-Phase Minisequencing 229Anu Suomalainen and Ann-Christine Syvanen

37 Nonradioactive Northern Blotting of RNA 239Rainer Low

38 Analysis of RNA by Northern Blotting Using Riboprobes 249Rai Ajit K. Srivastava

PART V GENE LIBRARY CONSTRUCTION AND SCREENING

39 Production of Double-Stranded cDNA for Gene Library Synthesis 261Jane Kirk and Steve Mayall

40 Using Rapid Amplification of cDNA Ends (RACE)to Obtain Full-Length cDNAs 267Yue Zhang and Michael A. Frohman

41 cDNA Library ConstructionUsing Streptavidin-Paramagnetic Beads and PCR 289Kris N. Lambert and Valerie M. Williamson

42 Rapid (Ligase-Free) Subcloningof Polymerase Chain Reaction Products 295A/an R. Shu/diner and Keith Tanner

43 Subtraction Hybridization cDNA Libraries 305Clifford W. Schweinfest, Peter S. Nelson, Michael W. Graber,Rita I. Demopoulos, and Takis S. Papas

44 Cloning Polymerase Chain Reaction ProductsUtilizing the T/A Overhang and a Kit 319Melissa Lail-Trecker

45 Extraction and Purification of Plasmid DNA 327Craig Winstanley and Ralph Rapley

46 Biotinylated Probes in Colony Hybridization 333Michael J. Haas

47 Cloning Long Polymerase Chain Reaction Products 339Songrong Ren and J. Michael Ruppert

48 Cloning DNA Fragments in M13 Vectors 347David Walsh

x Contents

49 cDNA Library Construction for the Lambda ZAP®-Based Vectors 355Marjory A. Snead, Michelle A. Alting-Mees, and Jay M. Short

50 Expression and Preparation of Fusion Proteinsfrom Recombinant )",gt11 Phages 367Sheng-He Huang and Ambrose Jong

51 Antibody Screening of Bacteriophage ,,-gt11 DNAExpression Libraries 373Peter Jones

52 Screening cDNA Libraries by Hybridizationwith Double-Stranded DNA Probes and Oligonucleotides 381Caroline A. Austin

53 cDNA Library Screening with the Tetramethylammonium Chloride (TMAC)Technique Using Highly Degenerate Oligoonucleotide Probes 389Bent Honore and Peder Madsen

54 Screening Recombinant Libraries by Polymerase Chain Reaction 397Michael W. King

55 Construction and Screening of Cosmid Libraries 405Jens Hanke and Jorg D. Hoheisel

56 Generation of Large Insert YAC Libraries 415Zoia Larin, Anthony P. Monaco, and Hans Lehrach

57 YAC Library Storage and Transport 425John E. Collins, Sheila Hassock, and Ian Dunham

58 YAC Library Screening: Preparation of Hybridization Filtersand Polymerase Chain Reaction Pools 431Charlotte G. Cole, John E. Collins, and Ian Dunham

59 YAC Library Screening: Hybridizationand PCR-Based Screening Protocols 437Charlotte G. Cole, John E. Collins, and Ian Dunham

60 Phage-Display Libraries of Murineand Human Antibody'Fab Fragments 449Jan Engberg, Lene K. Johansen, Michelle Westengaard-Hildinge,Erik S. Riise, and Bjarne Albrechtsen

PART VI DNA SEQUENCING

61 Preparation and Analysis of DNA Sequencing Gels 481Bimal D. M. Theophilus

62 DNA Sequencing of Plasmids 489George Murphy

63 Sequencing DNA Fragments Cloned into M13and Phagemid Vectors 493Neil Brewis

64 Direct cDNA Sequencing Using SequentialLinear/Asymmetric Polymerase Chain Reaction 499Ivor J. Mason

Contents xi

65 Purification and Enzymatic Sequencingof Polymerase Chain Reaction Products 505Frank C. Brosius III, Lawrence B. Holzman, and Xinan Cao

66 Direct Polymerase Chain Reaction Sequencingwith Denaturants 515Wei Zhang and Albert B. Deisseroth

67 Direct DNA Sequencing of Polymerase Chain Reaction ProductsUsing Magnetic Beads 523Joakim Lundeberg, Berti! Pettersson, and Mathias Uhlen

68 Polymerase Chain Reaction Cycle Sequencingwith Degenerate Primers 533Zhiyuan Shen, Jingmei Liu, Robert L. Wells,and Mortimer M. Elkind

69 Direct Automated Cycle Sequencingof Polymerase Chain Reaction Products 541Susan E. Daniels

70 Affinity-Capture and Solid-Phase Sequencingof Biotinylated Polymerase Chain Reaction Products 547Anu Suomalainen and Ann-Christine Syviinen

71 DNA Sequencing by the Chemical Method 553Eran Pichersky

72 One-Step One-Lane Chemical Sequencing of DNA 557Giovanna Costanzo, Ernesto Di Mauro, and Rodolfo Negri

PART VII BASIC POLYMERASE CHAIN REACTION METHODS

73 Polymerase Chain Reaction:Basic Principles and Routine Practice 569Lori A. Kolmodin and J. Fenton Williams

74 Primer Selection and Design for Polymerase Chain Reaction 581Wojciech Rychlik

75 One-Step Optimization Using Touchdown and StepdownPolymerase Chain Reaction 589Kenneth H. Roux and Karl H. Hecker

76 Cloning Gene Family Members Using Polymerase ChainReaction with Degenerate Oligonucleotide Primers 595Gregory M. Preston

77 Construction of Synthetic Genes by Polymerase Chain Reaction 609Patrick J. Dillon and Craig A. Rosen

78 Rapid Amplification of cDNA Ends 613David Bertioli

79 Multiplex Polymerase Chain Reaction 619Jerald Radich

80 Inverse Polymerase Chain Reaction 625Sheng-He Huang

xii Contents

81 Long Range Polymerase Chain Reaction 633William Waggott

PART VIII ANALYZING GENES, MUTATIONS, AND PROTEIN INTERACTIONS

82 Nonradioactive Differential Display of Messenger RNA 645Thomas C. G. Bosch and Jan U. Lohmann

83 Gene Isolation by Exon Trapping 653David B. Krizman

84 DNA Rescue by the Vectorette Method 667Marcia A. McAleer, Alison Coffey, and Ian Dunham

85 Random Amplified Polymorphic DNA (RAPDs) 675Scott Tingey

86 Restriction Fragment Length Polymorphism 679Mohammad S. Enayat

87 Detection of Mutations in DNA and RNA by Chemical Cleavage 685Richard G. H. Cotton

88 Mutation Screening Using PCR-SSCP:Silver Staining and Isotopic Protocols 695Philip J. Saker

89 Detecting Point Mutationsby Denaturing-Gradient Gel Electrophoresis 705Stephen R. Dlouhy, Patricia Wheeler, James A. Trofatter,Peter J. Stambrook, and Jay A. Tischfield

90 Analysis of Nucleotide Sequence Variationby Solid-Phase Minisequencing 717Anu Suomalainen and Ann-Christine Syvanen

91 The Amplification Refractory Mutation System 723John M. Old

92 DNase I Footprinting 729Benoit Leblanc and Tom Moss

93 Identification of Protein-DNA Contacts with Dimethyl Sulfate:Methylation Protection and Methylation Interference 737Peter E. Shaw and A. Francis Stewart

94 The Gel Shift Assay for the Analysis of DNA-Protein Interactions 745John D. Taylor, Alison J. Ackroyd, and Stephen E. Halford

95 Yeast Two-Hybrid Library Screening : 757Ian G. Cowell

96 The Southwestern Assay 773Jacques Philippe

97 Nonradioactive Methods for the Detectionof RNA-Protien Interaction 783Asier Echarri, Maria Eugenia Gonzalez, Ivan Ventoso,and Luis Carrasco

Contents xiii

98 Tanscriptional Activation Analysis by the ChloramphenicolAcetyl Transferase (CAT) Enzyme Assay 793David R. Hodge, Delores M. Thompson,Alexandra Panayiotakis, and Arun Seth

PART IX MUTAGENESIS, TRANSCRIPTION, AND TRANSLATION IN VITRO

99 Generating Nested Deletions with Exonuclease III 807George Murphy

100 Primer-Directed Site-Specific Mutagenesis 815Michael J. O'Donohue and G. Geoff Kneale

101 Site-Directed Mutagenesis Using a Uracil-ContainingPhagemid Template 827Christian Hagemeier

102 Site-Directed Mutagenesis Using Double-StrandedPlasmid DNA Templates 835Jeffrey Bramam, Carol Papworth, and Alan Greener

103 Site-Directed Mutagenesis with LA-PCRTM Technology 845Atsushi Shimada and Osamu Takeda

104 Recombination and Mutagenesisby Overlap Extension PCR 857Robert J. Pogulis, Abbe N. Vallejo, and Larry R. Pease

105 Site-Directed Mutagenesis and Gene Fusionby Megaprimer PCR 865Sailen Barik

106 Transcription In Vitro Using Bacteriophage RNA Polymerases 875Elaine T. Schenborn

107 In Vitro Translation of mRNAin a Rabbit Reticuloctye Lysate Cell-Free System 885Louise Olliver and Charles D. Boyd

108 In Vitro Translation of mRNAin a Wheat Germ Extract Cell-Free System 891Louise Olliver, Anne Grobler-Rabie, and Charles D. Boyd

109 The Xenopus Egg Extract Translation System 895Glenn M. Matthews and Alan Colman

110 Manipulation of Baculovirus Vectors 907Claire L. Merrington, Mark J. Bailey, and Robert D. Possee

111 Procedures for the Analysis and Purificationof His-Tagged Proteins 921Richard E. Kneusel, Joanne Crowe, Melanie Wulbeck,and Joachim Ribbe

112 Detection and Immobilization of ProteinsContaining the 6xHis Tag 935Richard E. Kneusel, Melanie Wulbeck, and Joachim Ribbe

xiv Contents

113 Expression and Purification of Recombinant ProteinsUsing the pET System 947Robert C. Mierendorf, Barbara B. Morris, Beth Hammer,and Robert E. Novy

PART X GENE LOCALIZATION, MAPPING IN SITU, AND BIOINFORMATICS

114 Preparation of Tissue Sections and Slidesfor mRNA Hybridization 981Giorgio Terenghi

115 Use of Digoxigenin-Labeled Probes on Tissue Sections 985Giorgio Terenghi

116 Gene Mapping by FISH 991Rafael Espinosa 11/ and Michelle M. Le Beau

117 Oligonucleotide PRINS DNA Synthesis 1011John R. Gosden and Diane Lawson

118 Chromosome-Specific PRINS 1017Jean-Paul Charlieu and Frank Pellestor

119 In Situ PCR Amplification of Intracellular mRNA 1023Raymond H. Chen and Susan V. Fuggle

120 An Introduction to Bioinformatics 1031Henry Brzeski

Index 1045

Contributors

ALISON J. ACKROYD • Department ofBiochemistry, University of TexasSouthwestern Medical Center, Dallas, TX

BJARNE ALBRECHTSEN • Department ofBiology, Royal Danish SchoolofPharmacy, Copenhagen, Denmark

MICHELLE A. ALTING-MEES • Stratagene Corporation, La Jolla, CASHU F. AN • Institute ofNeurology, University ofLondon, London, UKCAROLINE A. AUSTIN • Department ofBiochemistry and Genetics, The Medical

School, The University ofNewcastle upon Tyne, Newcastle, UKMARK 1. BAILEY • Institute of Virology and Environmental Microbiology, Oxford,

UKSAILEN BARIK • Department ofBiochemistry and Molecular Biology, College of

Medicine, University ofSouth Alabama, Mobile, ALDOMINIQUE BELIN· Department ofPathology, University of Geneva Medical Center,

Geneva, SwitzerlandDAVID BERTIOLI • Institute ofArable Crop Research, Rothampstead, Harpenden,

UKTHOMAS C. G. BOSCH • Institutfur Spezielle Zoologie, Universitat Jena, GermanyCHARLES D. BoYD • University ofMedicine and Dentistry, New Brunswick, NJJEFFREY BRAMAN • Stratagene, La Jolla, CANEIL BREWIS • Marie Curie Research Institute, Oxted, Surrey, UKFRANK C. BROSIUS III • Department ofInternal Medicine, Division

ofNephrology, University ofMichigan, Ann Arbor, MISIAN BRYANT • Tenovus Cancer Research Centre, University of Wales College of

Medicine, Cardiff, WalesHENRY BRZESKI • Department ofBiosciences, University ofHertfordshire, Hatfield,

UKXINAN CAO • Department ofInternal Medicine, Division ofNephrology, University

ofMichigan, Ann Arbor, MILUIS CARRASCO • Universidad Autonoma, Madrid, SpainELISABETH CHACHATY • Service de Microbiologie Medical, Institut Gustave-Roussy,

Villejuif FranceJEAN-PAUL CHARLlEU· Medical Research Council, Human Genetics Unit,

Western General Hospital, Edinburgh, UKRAYMOND H. CHEN· Department ofCardiac Surgery, Brigham and Women's Hospital,

Boston, MA

xv

xvi Contributors

SHu-HuA CHENG • Department ofMolecular Biology, Massachusetts GeneralHospital, Boston, MA

ALISON COFFEY • The Sanger Center, Wellcome Trust Genome Campus, Cambridge,UK

CHARLOTTE G. COLE· The Sanger Centre, Wellcome Trust Genome Campus,Cambridge, UK

ALAN COLMAN • Department ofSurgery, Queen Elizabeth Medical Center,Birmingham, UK

JOHN E. COLLINS • The Sanger Centre, Wellcome Trust Genome Campus, Cambridge,UK

GIOVANNA COSTANZO. Instituto Pasteur, Cenci Bolognetti, UniversitaLa Sapienza, Rome, Italy

RICHARD G. H. COTTON • Mutation Research Centre, St. Vincent's Hospital,Melbourne, Australia

IAN G. COWELL • Division ofIntegrative Biology, Roslin Institute (Edinburgh),Midlothian, Scotland, UK

JOANNE CROWE • Qiagen, Hilden, GermanyMARTIN W. CUNNINGHAM· Research and Development, Amersham Pharmacia

Biotech, Ltd., Buckinghamshire, UKSUSAN E. DANIELS • Sequana, La Jolla, San Diego, CAJANE DAVENPORT-JONES • Department ofBiosciences, University ofHertfordshire,

Hatfield, UKRITA I. DEMOPOULOS· DepCl;rtment ofPathology, New York University Medical

Center, New York, NYALBERT B. DEISSEROTH • School ofMedicine, Yale University, New Haven, CTPATRICK J. DILLON· Research and Development, Invitrogen, Carlsbad, CAERNESTO DI MAURO· Instituto Pasteur, Cenci Bolognetti, Universita

La Sapienza, Rome, ItalySTEPHEN R. DLOUHY • Department ofMedical Genetics, Indiana University

School ofMedicine, Indianapolis, INDDO DOBBELING • Department ofDermatology, University Hospital Zurich, Zurich,

SwitzerlandIAN DUNHAM • The Sanger Centre, Wellcome Trust Genome Campus, Cambridge,

UKIAN DURRANT • Research and Development, Amersham Pharmacia Biotech, Ltd.,

Buckinghamshire, UKASIER ECHARRI • Molecular Biology Department, University ofMadrid, Madrid,

SpainMORTIMER M. ELKIND • Department ofRadiological Health Sciences, Colorado State

University, Fort Collins, COMOHAMMAD S. ENAYAT • Department ofHaematology, Birmingham Children's

Hospital NHS Trust, Birmingham, UKJAN ENGBERG • Department ofBiology, Royal Danish School ofPharmacy,

Copenhagen, Denmark

Contributors xvii

RAFAEL ESPINOSA III • Section ofHematology/Oncology, DepartmentofMedicine, University ofChicago, Chicago, IL

PAOLA FERRO • Laboratory ofMolecular Biology, National Cancer Institute Genoa,Genoa, Italy

SUE FOWLER • Research and Development, Amersham Pharmacia Biotech, Ltd.,Buckinghamshire, UK

MICHAEL A. FROHMAN • Department ofPharmacology, University Medical Centerat Stony Brook, Stony Brook, NY

SUSAN V. FUGGLE • Nuffield Department ofSurgery, John Radcliffe Hospital,Oxford, UK

IAN GARNER • PPL Ltd., Edinburgh, ScotlandMARiA EUGENIA GONZALEZ • Universidad Autonoma, Madrid, SpainJOHN R. GOSDEN • MRC Human Genetics Unit, Western General Hospital,

Edinburgh, UK (Retired)MICHAEL W. GRABER • Department ofPathology and Laboratory Medicine, Albany

Medical Center Hospital, Albany, NYALAN GREENER • Stratagene, La Jolla, CAANNE GROBLER-RABIE· University ofMedicine and Dentistry, New Brunswick, NJMICHAEL J. HAAS • Eastern Regional Research Center, u.s. Department

ofAgriculture, Philadelphia, PACHRISTIAN HAGEMEIER • Laboratory ofMolecular Biology and Pediatric Disease,

Humboldt University, Charite Berlin, GermanySTEPHEN E. HALFORD • Centre for Molecular Recognition, University ofBristol,

Bristol, UKBETH HAMMER· Novagen, Madison, WIJENS HANKE· Functional Genome Analysis, Deutsches Krebsforschungszentrum, 1m

Neuenheimer Feld 506, Heidelberg, GermanyBRONWEN M. HARVEY • Research and Development, Amersham Pharmacia Biotech,

Ltd., Buckinghamshire, UKADRIAN J. HARWOOD • MRC Laboratory for Molecular Cell Biology, University

College London, London, UKSHEILA HASSOCK· Paediatric Research Unit, United Medical and Dental Schools

ofGuy's andSt. Thomas' Hospitals, London, UKKARL H. HECKER • Department ofBiological Sciences, Florida State University,

Tallahassee, FLJOHN HEPTINSTALL • NES-Biosciences, Coventry University, Coventry, UKDAVID R. HODGE • Laboratory ofMolecular Immunoregulation, Frederick Cancer

Research and Development Center, National Cancer Institute, Frederick, MDRACHEL HODGE • Department ofBotany, University ofLeicester, Leicester, UKJORG D. HOHEISEL • Functional Genome Analysis, Deutsches

Krebsforschungszentrum, 1m Neuenheimer Feld 506, Heidelberg, GermanyLAWRENCE B. HOLZMAN • Department ofInternal Medicine, Division

ofNephrology, University ofMichigan, Ann Arbor, MIBENT HONORE· Department ofMedical Biochemistry, University ofAarhus, Aarhus,

Denmark

xviii Contributors

SHENG-HE HUANG • Division ofInfectious Disease, Department ofPediatrics,University ofSouthern California, Children's Hospital ofLos Angeles, LosAngeles, CA

PETER G. ISAAC • Nickersen-Biochem, Cambridge, UKLENE K. JOHANSEN· Department ofBiology, Royal Danish School

ofPharmacy, Copenhagen, DenmarkPETER JONES • School ofBiomedical Sciences, University ofNottingham, Queen's

Medical Centre, Nottingham, UKAMBROSE JONG • Division ofInfectious Disease, Department ofPediatrics, University

ofSouthern California, Children's Hospital ofLos Angeles, Los Angeles, CAELENA D. KATZ • Department ofBiotechnology, Perkin Elmer, Norwalk, CTROSEMARY KELSELL • Formerly Department ofMolecular Genetics, Institute of

Ophthalmology, London, UKMICHAEL W. KING • Department ofBiochemistry and Molecular Biology, School

ofMedicine, Indiana University, Terre Haute, INJANE KIRK • ICRF, Clare Hall Laboratories, South Mimms, Herts, UKG. GEOFF KNEALE • Biophysics Laboratories, University ofPortsmouth, Portsmouth,

UKRICHARD E. KNEUSEL • Qiagen, Hilden, GermanyLORI A, KOLMODIN • Roche Molecular Systems, Alameda, CADAVID B. KRIZMAN • Laboratory ofPathology, National Insitutes ofHealth.

Bethesda, MDMELISSA LAIL-TRECKER • Department ofAnatomy, The School ofMedicine,

University ofConnecticut Health Center, Farmington, CTKRIS N. LAMBERT • Department ofHematology, University ofCalifornia, Davis, CAZOIA LARIN • Institute ofMolecular Medicine, University ofOxford,

Oxford, UKDIANE LAWSON • MRC Human Genetics Unit, Western General Hospital, Edinburgh,

UKMICHELLE M. LE BEAU • Section ofHaematology/Oncology, Department

ofMedicine, University ofChicago, Chicago, ILBENOiT LEBLANC • Centre de Recherche en Cancerologie et Departement

de Biologie Medicale de I'Universite Laval, Centre Hopital Universitaire deQuebec (CHUQ), Pavillon H6tel-Dieu de Quebec, Quebec, Canada

HANS LEHRACH • Max Planck Institt.t fur Moleculare Genetik, Berlin, GermanyMARK LEONARD • Developmental Biology Research Centre, Kings College,

University ofLondon, London, UKJINGMEI LIU • Department ofRadiological Health Sciences, Colorado State

University, Fort Collins, COY. M. DENNIS Lo· Department ofChemical Pathology, Prince of Wales Hospital,

The Chinese University ofHong Kong, Hong Kong, People's Republic ofChinaJAN U. LOHMANN • Salk Institute, La Jolla, CARAINER Low • Zentrum Moleculare Biologie Heidelberg (ZMBH), Heidelberg,

Germany

Contributors xix

JOAKIM LUNDEBERG • Department ofBiochemistry, Kungliga Tekniska Hogskolan,Royal Institute of Technology, Stockholm, Sweden

PEDER MADSEN • Department ofMedical Biochemistry, University ofAarhus,Aarhus, Denmark

FRAN<;:OIS MALLET • CNRS-BioMerieux, Lyon, FranceDAVID L. MANNING • Tenovus Cancer Research Centre, University of Wales College

ofMedicine, Cardiff, Wales, UKROBERTO MANTOVANI • Departimento de Genetica e di Biologia dei Microrganismi,

Universita de Milano, Milan, ItalyIVOR J. MASON • Department ofDevelopmental Neurobiology, King's College

London, Guy's Hospital Campus, London, UKGLENN M. MATTHEWS· Department ofSurgery, Queen Elizabeth Medical Centre,

Birmingham, UKJOHN MAULE • MRC Human Genetics Unit, Molecular Genetics Section, Western

General Hospital, Edinburgh, Scotland, UKSTEVE MAYALL • ICRF, Clare Hall Laboratories, South Mimms, Herts, UKMARCIA A. McALEER· Yamanouchi Research Institute, Littlemore Park, Oxford, UKCLAIRE L. MERRINGTON • Institute of Virology and Environmental Microbiology,

Oxford, UKROBERT C. MIERENDORF • Novagen, Madison, WIANTHONY P. MONACO • Wellcome Trust Centre for Human Genetics, University

ofOxford, Oxford, UKBRANDON D. MOORE • Department ofBiochemistry, University ofNevada, Reno, NVBARBARA B. MORRIS • Novagen, Madison, WITOM Moss· Centre de Recherche en Cancerologie et Departement de Biologie

Medicale de l'Universite Laval, Centre Hopital Universitaire de Quebec(CHUQ), Pavillon Hotel-Dieu de Quebec, Quebec, Canada

GEORGE MURPHY· John Innis Centrefor Plant Science Research, Norwich, UKRODOLFO NEGRI· Centro Acidi Nucleici, CNR, ROME, hALY

PETER S. NELSON • Department ofMolecular Biotechnology, Universityof Washington, Seattle, WA

ROBERT E. Novy· Novagen, Madison, WIMICHAEL J. O'DONOHUE • UBPB-Fractionnement Enzymatique, INRA-Lille/Reims,

Reims, FranceJOHN M. OLD • Institute ofMolecular Medicine, John Radcliffe Hospital,

Headington, Oxford, UKLOUISE OLLIVER • University ofMedicine and Dentistry, New Brunswick, NJALEXANDRA PANAYIOTAKIS • Laboratory ofMolecular Oncology National Cancer

Institute, Frederick, MDTAKIS S. PAPAS • Center for Molecular and Structural Biology, Medical University

ofSouth Carolina, Charleston, SCCAROL P APWORTH • Stratagene, La Jolla, CAROGER PATIENT • Developmental Biology Research Centre, Kings College,

University ofLondon, London

xx Contributors

LARRY R. PEASE • Department ofImmunology, Mayo Clinic and Mayo Foundation,Rochester, MN

FRANK PELLESTOR • Medical Research Council, Human Genetics Unit, WesternGeneral Hospital, Edinburgh, UK

BERTIL PETTERSSON • Department ofBiochemistry, Kungliga Tekniska Hogskolan,Royal Institute of Technology, Stockholm, Sweden

JACQUES PHILIPPE • Diabetes Unit, Department ofMedicine, Centre MedicalUniversitaire, Geneva, Switzerland

ULRICH PFEFFER • Laboratory ofMolecular Biology, National Cancer InstituteGenoa, Genoa, Italy

ERAN PICHERSKY • Department ofBiology, Natural Sciences Building, UniversityofMichigan, Ann Arbor, MI

ROBERT 1. POGULIS • Department ofImmunology, Mayo Clinic and MayoFoundation, Rochester, MN

ROBERT D. POSSEE • Institute of Virology and Environmental Microbiology, Oxford,UK

GREGORY M. PRESTON • Department ofMolecular Sciences, Central ResearchDivision, Pfizer, Inc., Groton, CT

ERIC QUEMENEUR· Departement d'Ingenierie et d'Etudes des Proteines,CEA, Saclay, France

JERALD RADICH • Division ofClinical Research, Fred Hutchinson Cancer Center,Seattle, WA

RALPH RAPLEY • Department ofBiosciences, University ofHertfordshire, Hatfield,UK

SONGRONG REN • Department ofMedicine, University ofAlabama, Birmingham, ALRALF RESKI • Freiburg University Institute Biology II, Frieburg/Breisgua, Germany

OR Institute for General Botany, Hamburg, GermanyJOACHIM RIBBE • Qiagen, Hilden, GermanyERIK S. RIISE • Department ofBiology, Royal Danish School ofPharmacy,

Copenhagen, DenmarkCRAIG A. ROSEN· Research and Development, Human Genome Sciences, Rockville,

MDKENNETH H. Roux • Department ofBiological Sciences, Florida State University,

Tallahassee, FL1. MICHAEL RUPPERT • Department ofMedicine, University ofAlabama, Birmingham,

ALWOJCIECH RYCHLIK • National Biosciences, Plymouth, MNPHILIP 1. SAKER • Complex Traits Analysis Group, Imperial College School

ofMedicine, Hammersmith Hospital, London, UKPATRICK SAULNIER • Service de Microbiologie Medical, Institut

Gustave-Roussy, Villejuif, FranceELAINE T. SCHENBORN • Promega Corporation, Madison, WIGUSTAV SCHONFELD· Department ofInternal Medicine, Washington University

School ofMedicine, St. Louis, MO

Contributors xxi

CLIFFORD W. SCHWEINFEST • Center for Molecular and Structural Biology, MedicalUniversity ofSouth Carolina, Charleston, SC

JEFFREY R. SEEMANN • Department ofBiochemistry, University ofNevada, Reno, NVARUN SETH • Laboratory ofMolecular Oncology National Cancer Institute,

Frederick, MDPETER E. SHAW • School ofBiomedical Sciences, University ofNottingham, Queen's

Medical Centre, Nottingham, UKZHIYUAN SHEN • Department ofMolecular Genetics, University ofIllinois

at Chicago, Chicago, ILATSUSHI SHIMADA • Shuzo Co., Ltd., Otsu, Shiga, JapanJAY M. SHORT • Diversa Corporation, San Diego, CAALAN R. SHULDINER • Johns Hopkins University Schopl ofMedicine, Baltimore, MDDUNCAN R. SMITH· Molecular Biology Laboratory, Tan Tock Seng Hospital, SingaporeMARJORY A. SNEAD· Diversa Corporation, San Diego, CASURAPAREDDY SREENIVASAPRASAD • Department ofPlant Pathology and Microbiology,

Horticulture Research International, Wellesbourne, Warwickshire, UKRAJ Am K. SRIVASTAVA· Department ofInternal Medicine, Washington University

School ofMedicine, St. Louis, MOJUSTIN STACEY· Biogemma, UK, Ltd., Cambridge, UKPETER 1. STAMBROOK • Department ofMedical Genetics, Indiana School

ofMedicine, Indianapolis, INA. FRANCIS STEWART • School ofBiomedical Sciences, University ofNottingham,

Queen's Medical Centre, Nottingham, UKTIMOTHY STONE • Resarch and Development, Amersham Pharmacia Biotech, Ltd.,

Buckinghamshire, UKANU SUOMALAINEN • National Public Health Institute, Department ofHuman

Molecular Genetics, Helsinki, FinlandANN-CHRISTINE SYVANEN • Department ofMedical Science and Molecular Medicine,

Uppsala University Hospital, Uppsala, SwedenOSAMU TAKEDA· Shuzo Co., Ltd., Kusatsu, JapanKEITH TANNER • Johns Hopkins University School ofMedicine,

Baltimore, MDJOHN D. TAYLOR· Department ofBiochemistry, University ofTexas Southwestern

Medical Center, Dallas, TXGIORGIO TERENGHI • Blond McIndoe Laboratories, University Department

ofSurgery, Royal Free and University College Medical School, UniversityCollege London, London, UK

DELORES M. THOMPSON • Laboratory ofMolecular Oncology, National CancerInstitute, Frederick, MD

BIMAL D. M. THEOPHILUS • Department ofHaematology, Birmingham Children'sHospital NHS Trust, Birmingham, UK

SCOTT TINGEY· DuPont Agricultural Products, Wilmington, DEJAY A. TISCHFIELD • Department ofMedical Genetics, Indiana School

ofMedicine, Indianapolis, IN

xxii Contributors

JAMES A. TROFATTER • Department ofMedical Genetics, Indiana SchoolofMedicine, Indianapolis, IN

MATHIAS UHLEN • Department ofBiochemistry, Kungliga Tekniska Hogskolan, RoyalInstitute ofTechnology, Stockholm, Sweden

ABBE N. VALLEJO • Department ofImmunology, Mayo Clinic and Mayo Foundation,Rochester, MN

IvAN VENTOSO • Universidad Autonoma, Madrid, SpainSTEPHANE VIVILLE • Institut de Gimetique et de Biologie Moleculaire et Cellulaire,

CNRSIINSERMIULP, Strasbourg, FranceWILLIAM WAGGOTT • Department ofCellular Sciences, John Radcliffe Hospital,

University ofOxford, UKMAGGIE WALMSLEY • Developmental Biology Research Centre, Kings College,

University ofLondon, London, UKDAVID WALSH • Scottish Agricultural College, Edinburgh, ScotlandROBERT L. WELLS • Department ofRadiological Health Sciences, Colorado State

University, Fort Collins, COMICHELLE WESTENGAARD-HILDINGE • Department ofBiology, Royal Danish School

ofPharmacy, Copenhagen, DenmarkCLAIRE B. WHEELER • Research and Development, Amersham Pharmacia Biotech,

Ltd., Buckinghamshire, UKPATRICIA WHEELER· Department ofMedical Genetics, Indiana School

ofMedicine, .Indianapolis, IN1. FENTON WILLIAMS • PE Biosystems, Foster City, CAD. Ross WILLIAMS • Department ofBiosciences, University ofHertfordshire,

Hatfield, UKVALERIE M. WILLIAMSON • Department ofHematology, University

ofCalifornia, Davis, CACRAIG WINSTANLEY • Depart"'!ent ofMedical Microbiology, University

ofLiverpool, Liverpool, UKMELANIE WULBECK • Qiagen, Hilden, GermanyFEI YE • Department ofBiology, Massachusetts Institute of Technology, Cambridge,

MAWEI ZHANG • Departments ofPathology and Neuro-Oncology, The University

ofTexas M. D. Anderson Cancer Center, Houston, TXYUE ZHANG • Department ofPharmacology, University Medical Center

at Stony Brook, Stony Brook, NY