THE NUCLEIC ACID PROTOCOLS HANDBOOK - Home - …978-1-59259-0… · · 2017-08-25Justin...
Transcript of THE NUCLEIC ACID PROTOCOLS HANDBOOK - Home - …978-1-59259-0… · · 2017-08-25Justin...
THE NUCLEIC ACID PROTOCOLS HANDBOOK
TheNucleic Acid
ProtocolsHandbook
Edited by
Ralph RapleyUniversity ofHertfordshire
HatfieldUK
Humana Press *- Totowa, New Jersey
© 2000 Humana Press Inc.999 Riverview Drive, Suite 208Totowa, New Jersey 07512
All rights reserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted in any formor by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise without written permission from the Publisher. Methods in Molecular Biology'" is a trademark of The Humana Press Inc.
The content and opinions expressed ih this book are the sole work of the authors and editors, who have warranted duediligence in the creation and issuance of their work. The publisher, editors, and authors are not responsible for errorsor omissions or for any consequences arising from the information or opinions presented in this book and make nowarranty, express or implied, with respect to its contents.
This publication is printed on acid-free paper. @
ANSI Z39.48-1984 (American Standards Institute) Permanence of Paper for Printed Library Materials.
Cover design by Patricia F. Cleary.
For additional copies, pricing for bulk purchases, andlor information about other Humana titles, contact Humana at theabove address or at any of the following numbers: Tel: 973-256-1699; Fax: 973-256-8341; E-mail:[email protected], or visit our Website at www.humanapress.com
Photocopy Authorization Policy:
Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients, isgranted by Humana Press Inc., provided that the base fee of US $10.00 per copy, plus US $00.25 per page, is paiddirectly to the Copyright Clearance Center at 222 Rosewood Drive, Danvers, MA 01923. For those organizations thathave been granted a photocopy license from the CCC, a separate system ofpayment has been arranged and is acceptableto Humana Press Inc. The fee code for users of the Transactional Reporting Service is: [0-89603-459-3/00 $10.00 +$00.25].
Printed in the United States of America. 10 9 8 7 6 5 4 3 2
Library of Congress Cataloging in Publication Data
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