THE NUCLEIC ACID PROTOCOLS HANDBOOK

The
Nucleic Acid
Protocols
Handbook
Edited by
Ralph Rapley
University ofHertfordshire
Hatfield
*-
2000 Humana Press Inc.
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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 edited by Ralph Rapley.
p. em.
Includes index.
ISBN 0-89603-459-3 (alk. paper) (hardcover), ISBN 0-89603-841-6 (paper)
I. Nucleic manuals. 2. Polymerase chain manu-
als. 3. Gene manuals. 4. Molecular manuals.
Rapley, Ralph.
QP620.N7987 1999
572.8---dc21 98-43385
CIP
There can be no doubt that some of the most spectacular advances made in science
over the past few decades have been in the isolation, analysis, and manipulation of
nucleic acids. This has led to a much greater understanding ofmechanisms and processes
across 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 of
the biotechnology industry, which seeks to develop and commercialize many of these
important processes and methods. Much of this has come about because of the devel-
opment of numerous molecular biology and genetic manipulation techniques. The
discovery of restriction enzymes and the development of cloning vectors in the early
1970s opened the door to ways ofisolating and manipulating nucleic acids that had never
been thought possible. Gene probe labeling and hybridization were developed and
refined to provide powerful methods ofanalysis. These-together with the development
of DNA sequencing methods, protein engineering techniques, and PeR-have all
continued to contribute substantially to the understanding ofbiological processes at the
molecular level. The protocols for these important methods are the focus of The Nucleic
Acid Protocols Handbook, whose aim is to provide a comprehensive set of techniques
in one volume that will enable the isolation, analysis, and manipulation ofnucleic acids
to be readily undertaken.
The Nucleic Acid Protocols Handbook is divided into 10 parts; within each there are
approximately 10 chapters. The first four parts follow one another logically: nucleic acid
extraction (Part I), basic separation and analysis of DNA (II), through probe design and
labeling (III), and RNA analysis techniques (IV). The following three sections deal with
gene library construction and screening (V), DNA sequencing (VI), and the polymerase
chain reaction (VII). Part VIII deals with the analysis of genes, mutations, and protein
interactions and is followed by Part IX, on mutagenesis, transcription, and translation in
vitro. 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 from
versions appearing in earlier volumes of Humana Press' Methods in Molecular Biology
series. These highly successful books have provided numerous laboratories with the
techniques needed to undertake modern laboratory molecular biology success
fully. As such, their format has been followed in The Nucleic AcidProtocols Handbook.
Thus a short introduction to the basic theory ofthe technique is followed by a complete
listing 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 are
cited throughout the Methods and appear at the end of the chapter, providing valuable
and highly useful information not found in traditional scientific literature. This essential
v
infonnation in many cases may mean the difference between the success or failure of
a particular technique and is one of the recognized key points of the Humana Methods
in 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 element
of many of the protocols. These have been cross referenced where possible, although
most of the protocols are self-contained and can be attempted without the need to read
further chapters. For those new or unfamiliar to laboratory molecular biology, the
compilation of protocols in The Nucleic Acid Protocols Handbook also provides the
ability to attempt protocols confidently. The intent was not to list all protocols in molec-
ular biology (within one volume, this is an impossible task), and certainly more
advanced protocols may be found in a number of excellent texts including many in the
Humana Methods in Molecular Biology series. was, however, the aim to provide the
most commonly used protocols and alternatives in one volume at a level accessible to
most 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 write
and update these protocols; to Prof. John M. Walker, the series editor, for his helpful
advice and guidance; and to the staffat Humana Press for their substantial efforts in the
production of the volume.
Ralph Rapley
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875
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891
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921
C.
IN
120
Department ofBiochemistry, University of Texas
Southwestern Medical Center, Dallas, TX
Department ofBiology, Royal Danish School
ofPharmacy, Copenhagen, Denmark
Stratagene Corporation, La Jolla, CA
Institute ofNeurology, University ofLondon, London, UK
Department ofBiochemistry and Genetics, The Medical
School, The University ofNewcastle upon Tyne, Newcastle, UK
1. Institute of Virology and Environmental Microbiology, Oxford,
UK
Department ofBiochemistry and Molecular Biology, College of
Medicine, University ofSouth Alabama, Mobile, AL
Department ofPathology, University of Geneva Medical Center,
Geneva, Switzerland
Institute ofArable Crop Research, Rothampstead, Harpenden,
UK
C. G. Institutfur Spezielle Zoologie, Universitat Jena, Germany
University ofMedicine and Dentistry, New Brunswick, NJ
Stratagene, La Jolla, CA
Marie Curie Research Institute, Oxted, Surrey, UK
III Department ofInternal Medicine, Division
ofNephrology, University ofMichigan, Ann Arbor, MI
Tenovus Cancer Research Centre, University of Wales College of
Medicine, Cardiff, Wales
Department ofBiosciences, University ofHertfordshire, Hatfield,
UK
Department ofInternal Medicine, Division ofNephrology, University
ofMichigan, Ann Arbor, MI
Universidad Autonoma, Madrid, Spain
Service de Microbiologie Medical, Institut Gustave-Roussy,
Villejuif France
Medical Research Council, Human Genetics Unit,
Western General Hospital, Edinburgh, UK
Department ofCardiac Surgery, Brigham and Women's Hospital,
Boston, MA
xv
Department ofMolecular Biology, Massachusetts General
Hospital, Boston, MA
The Sanger Center, Wellcome Trust Genome Campus, Cambridge,
UK
The Sanger Centre, Wellcome Trust Genome Campus,
Cambridge, UK
Department ofSurgery, Queen Elizabeth Medical Center,
Birmingham, UK
The Sanger Centre, Wellcome Trust Genome Campus, Cambridge,
UK
Instituto Pasteur, Cenci Bolognetti, Universita
La Sapienza, Rome, Italy
Mutation Research Centre, St. Vincent's Hospital,
Melbourne, Australia
Division ofIntegrative Biology, Roslin Institute (Edinburgh),
Midlothian, Scotland, UK
Qiagen, Hilden, Germany
W. Research and Development, Amersham Pharmacia
Biotech, Ltd., Buckinghamshire, UK
Sequana, La Jolla, San Diego, CA
Department ofBiosciences, University ofHertfordshire,
Hatfield, UK
DepCl;rtment ofPathology, New York University Medical
Center, New York, NY
B. School ofMedicine, Yale University, New Haven, CT
Research and Development, Invitrogen, Carlsbad, CA
Instituto Pasteur, Cenci Bolognetti, Universita
La Sapienza, Rome, Italy
R. Department ofMedical Genetics, Indiana University
School ofMedicine, Indianapolis, IN
Department ofDermatology, University Hospital Zurich, Zurich,
Switzerland
The Sanger Centre, Wellcome Trust Genome Campus, Cambridge,
UK
Research and Development, Amersham Pharmacia Biotech, Ltd.,
Buckinghamshire, UK
Molecular Biology Department, University ofMadrid, Madrid,
Spain
Department ofRadiological Health Sciences, Colorado State
University, Fort Collins, CO
Department ofHaematology, Birmingham Children's
Hospital NHS Trust, Birmingham, UK
Department ofBiology, Royal Danish School ofPharmacy,
Copenhagen, Denmark
RAFAEL ESPINOSA Section ofHematology/Oncology, Department
ofMedicine, 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 Center
at Stony Brook, Stony Brook, NY
SUSAN FUGGLE Nuffield Department ofSurgery, John Radcliffe Hospital,
Oxford, UK
IAN GARNER PPL Ltd., Edinburgh, Scotland
MARiA EUGENIA GONZALEZ Universidad Autonoma, Madrid, Spain
JOHN 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, NY
ALAN GREENER Stratagene, La Jolla, CA
ANNE GROBLER-RABIE University ofMedicine and Dentistry, New Brunswick, NJ
MICHAEL J. HAAS Eastern Regional Research Center, u.s. Department
ofAgriculture, Philadelphia, PA
CHRISTIAN HAGEMEIER Laboratory ofMolecular Biology and Pediatric Disease,
Humboldt University, Charite Berlin, Germany
STEPHEN E. HALFORD Centre for Molecular Recognition, University ofBristol,
Bristol, UK
BETH HAMMER Novagen, Madison, WI
JENS HANKE Functional Genome Analysis, Deutsches Krebsforschungszentrum, 1m
Neuenheimer Feld 506, Heidelberg, Germany
BRONWEN M. HARVEY Research and Development, Amersham Pharmacia Biotech,
Ltd., Buckinghamshire, UK
ADRIAN J. HARWOOD MRC Laboratoryfor Molecular Cell Biology, University
College London, London, UK
SHEILA HASSOCK Paediatric Research Unit, United Medical and Dental Schools
of Guy's andSt. Thomas' Hospitals, London, UK
KARL H. HECKER Department ofBiological Sciences, Florida State University,
Tallahassee, FL
JOHN HEPTINSTALL NES-Biosciences, Coventry University, Coventry, UK
DAVID R. HODGE Laboratory ofMolecular Immunoregulation, Frederick Cancer
Research and Development Center, National Cancer Institute, Frederick, MD
RACHEL HODGE Department ofBotany, University ofLeicester, Leicester, UK
JORG D. HOHEISEL Functional Genome Analysis, Deutsches
Krebsforschungszentrum, 1m Neuenheimer Feld 506, Heidelberg, Germany
LAWRENCE B. HOLZMAN Department ofInternal Medicine, Division
ofNephrology, University ofMichigan, Ann Arbor, MI
BENT HONORE Department ofMedical Biochemistry, University ofAarhus, Aarhus,
Denmark
SHENG-HE HUANG Division ofInfectious Disease, Department ofPediatrics,
University ofSouthern California, Children's Hospital ofLos Angeles, Los
Angeles, CA
PETER G. ISAAC Nickersen-Biochem, Cambridge, UK
LENE K. JOHANSEN Department ofBiology, Royal Danish School
ofPharmacy, Copenhagen, Denmark
PETER JONES School ofBiomedical Sciences, University ofNottingham, Queen's
Medical Centre, Nottingham, UK
AMBROSE JONG Division ofInfectious Disease, Department ofPediatrics, University
ofSouthern California, Children's Hospital ofLos Angeles, Los Angeles, CA
ELENA D. KATZ Department ofBiotechnology, Perkin Elmer, Norwalk, CT
ROSEMARY KELSELL Formerly Department ofMolecular Genetics, Institute of
Ophthalmology, London, UK
MICHAEL W. KING Department ofBiochemistry and Molecular Biology, School
ofMedicine, Indiana University, Terre Haute, IN
JANE KIRK ICRF, Clare Hall Laboratories, South Mimms, Herts, UK
G. GEOFF KNEALE Biophysics Laboratories, University ofPortsmouth, Portsmouth,
UK
RICHARD E. KNEUSEL Qiagen, Hilden, Germany
LORI A, KOLMODIN Roche Molecular Systems, Alameda, CA
DAVID B. KRIZMAN Laboratory ofPathology, National Insitutes ofHealth.
Bethesda, MD
MELISSA LAIL- TRECKER Department ofAnatomy, The School ofMedicine,
University ofConnecticut Health Center, Farmington, CT
KRIS N. LAMBERT Department ofHematology, University ofCalifornia, Davis, CA
ZOIA LARIN Institute ofMolecular Medicine, University ofOxford,
Oxford, UK
DIANE LAWSON MRC Human Genetics Unit, Western General Hospital, Edinburgh,
UK
MICHELLE M. LE BEAU Section ofHaematology/Oncology, Department
ofMedicine, University ofChicago, Chicago, IL
BENOiT LEBLANC Centre de Recherche en Cancerologie et Departement
de Biologie Medicale de I'Universite Laval, Centre Hopital Universitaire de
Quebec (CHUQ), Pavillon H6tel-Dieu de Quebec, Quebec, Canada
HANS LEHRACH Max Planck Institt.t fur Moleculare Genetik, Berlin, Germany
MARK LEONARD Developmental Biology Research Centre, Kings College,
University ofLondon, London, UK
JINGMEI LIU Department ofRadiological Health Sciences, Colorado State
University, Fort Collins,
Y. M. DENNIS Lo Department ofChemical Pathology, Prince of Wales Hospital,
The Chinese University ofHong Kong, Hong Kong, People's Republic ofChina
JAN U. LOHMANN Salk Institute, La Jolla, CA
RAINER Low Zentrum Moleculare Biologie Heidelberg (ZMBH), Heidelberg,
Germany
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, France
DAVID MANNING Tenovus Cancer Research Centre, University of Wales College
ofMedicine, Cardiff, Wales, UK
ROBERTO MANTOVANI Departimento de Genetica e di Biologia dei Microrganismi,
Universita de Milano, Milan, Italy
IVOR J. MASON Department ofDevelopmental Neurobiology, King's College
London, Guy's Hospital Campus, London, UK
GLENN M. MATTHEWS Department ofSurgery, Queen Elizabeth Medical Centre,
Birmingham, UK
JOHN MAULE MRC Human Genetics Unit, Molecular Genetics Section, Western
General Hospital, Edinburgh, Scotland, UK
STEVE MAYALL ICRF, Clare Hall Laboratories, South Mimms, Herts, UK
MARCIA McALEER Yamanouchi Research Institute, Littlemore Park, Oxford, UK
CLAIRE MERRINGTON Institute of Virology and Environmental Microbiology,
Oxford, UK
ROBERT C. MIERENDORF Novagen, Madison, WI
ANTHONY P. MONACO Wellcome Trust Centre for Human Genetics, University
ofOxford, Oxford, UK
BRANDON D. MOORE Department ofBiochemistry, University ofNevada, Reno, NV
BARBARA B. MORRIS Novagen, Madison, WI
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, UK
RODOLFO NEGRI Centro Acidi Nucleici, CNR, ROME, hALY
PETER S. NELSON Department ofMolecular Biotechnology, University
of Washington, Seattle, WA
ROBERT E. Novy Novagen, Madison, WI
MICHAEL J. O'DONOHUE UBPB-Fractionnement Enzymatique, INRA-Lille/Reims,
Reims, France
JOHN M. OLD Institute ofMolecular Medicine, John Radcliffe Hospital,
Headington, Oxford, UK
LOUISE OLLIVER University ofMedicine and Dentistry, New Brunswick, NJ
ALEXANDRA PANAYIOTAKIS Laboratory ofMolecular Oncology National Cancer
Institute, Frederick, MD
TAKIS S. PAPAS Centerfor Molecular and Structural Biology, Medical University
ofSouth Carolina, Charleston,
CAROL P APWORTH Stratagene, La Jolla, CA
ROGER PATIENT Developmental Biology Research Centre, Kings College,
University ofLondon, London
Department ofImmunology, Mayo Clinic and Mayo Foundation,
Rochester, MN
Medical Research Council, Human Genetics Unit, Western
General Hospital, Edinburgh, UK
Department ofBiochemistry, Kungliga Tekniska Hogskolan,
Royal Institute of Technology, Stockholm, Sweden
Diabetes Unit, Department ofMedicine, Centre Medical
Universitaire, Geneva, Switzerland
Laboratory ofMolecular Biology, National Cancer Institute
Genoa, Genoa, Italy
Department ofBiology, Natural Sciences Building, University
ofMichigan, Ann Arbor, MI
Department ofImmunology, Mayo Clinic and Mayo
Foundation, Rochester, MN
D. Institute of Virology and Environmental Microbiology, Oxford,
UK
Department ofMolecular Sciences, Central Research
Division, Pfizer, Inc., Groton, CT
Departement d'Ingenierie et d'Etudes des Proteines,
CEA, Saclay, France
Division ofClinical Research, Fred Hutchinson Cancer Center,
Seattle, WA
Department ofBiosciences, University ofHertfordshire, Hatfield,
UK
Department ofMedicine, University ofAlabama, Birmingham, AL
Freiburg University Institute Biology Frieburg/Breisgua, Germany
OR Institute for General Botany, Hamburg, Germany
Qiagen, Hilden, Germany
Department ofBiology, Royal Danish School ofPharmacy,
Copenhagen, Denmark
Research and Development, Human Genome Sciences, Rockville,
MD
Department ofBiological Sciences, Florida State University,
Tallahassee, FL
Department ofMedicine, University ofAlabama, Birmingham,
AL
National Biosciences, Plymouth, MN
Complex Traits Analysis Group, Imperial College School
ofMedicine, Hammersmith Hospital, London, UK
Service de Microbiologie Medical, Institut
Gustave-Roussy, Villejuif, France
T. Promega Corporation, Madison, WI
Department ofInternal Medicine, Washington University
School ofMedicine, St. Louis, MO
W. Center for Molecular and Structural Biology, Medical
University ofSouth Carolina, Charleston,
Department ofBiochemistry, University ofNevada, Reno, NV
Laboratory ofMolecular Oncology National Cancer Institute,
Frederick, MD
School ofBiomedical Sciences, University ofNottingham, Queen's
Medical Centre, Nottingham, UK
Department ofMolecular Genetics, University ofIllinois
at Chicago, Chicago, IL
Shuzo Co., Ltd., Otsu, Shiga, Japan
Diversa Corporation, San Diego, CA
Johns Hopkins University Schopl ofMedicine, Baltimore, MD
Molecular Biology Laboratory, Tan Tock Seng Hospital, Singapore
Diversa Corporation, San Diego, CA
Department ofPlant Pathology and Microbiology,
Horticulture Research International, Wellesbourne, Warwickshire, UK
Department ofInternal Medicine, Washington University
School ofMedicine, St. Louis, MO
Biogemma, UK, Ltd., Cambridge, UK
1. Department ofMedical Genetics, Indiana School
ofMedicine, Indianapolis, IN
School ofBiomedical Sciences, University ofNottingham,
Queen's Medical Centre, Nottingham, UK
Resarch and Development, Amersham Pharmacia Biotech, Ltd.,
Buckinghamshire, UK
National Public Health Institute, Department ofHuman
Molecular Genetics, Helsinki, Finland
Department ofMedical Science and Molecular Medicine,
Uppsala University Hospital, Uppsala, Sweden
Shuzo Co., Ltd., Kusatsu, Japan
Johns Hopkins University School ofMedicine,
Baltimore, MD
Department ofBiochemistry, University of Texas Southwestern
Medical Center, Dallas, TX
Blond McIndoe Laboratories, University Department
ofSurgery, Royal Free and University College Medical School, University
College London, London, UK
Laboratory ofMolecular Oncology, National Cancer
Institute, Frederick, MD
Department ofHaematology, Birmingham Children's
Hospital NHS Trust, Birmingham, UK
DuPont Agricultural Products, Wilmington, DE
Department ofMedical Genetics, Indiana School
ofMedicine, Indianapolis, IN
Department ofMedical Genetics, Indiana School
ofMedicine, Indianapolis, IN
Department ofBiochemistry, Kungliga Tekniska Hogskolan, Royal
Institute of Technology, Stockholm, Sweden
Department ofImmunology, Mayo Clinic and Mayo Foundation,
Rochester, MN
Universidad Autonoma, Madrid, Spain
Institut de Gimetique et de Biologie Moleculaire et Cellulaire,
CNRSIINSERMIULP, Strasbourg, France
Department ofCellular Sciences, John Radcliffe Hospital,
University ofOxford, UK
Developmental Biology Research Centre, Kings College,
University ofLondon, London, UK
Scottish Agricultural College, Edinburgh, Scotland
Department ofRadiological Health Sciences, Colorado State
University, Fort Collins,
Department ofBiology, Royal Danish School
ofPharmacy, Copenhagen, Denmark
B. Research and Development, Amersham Pharmacia Biotech,
Ltd., Buckinghamshire, UK
Department ofMedical Genetics, Indiana School
ofMedicine, .Indianapolis, IN
1. PE Biosystems, Foster City, CA
Department ofBiosciences, University ofHertfordshire,
Hatfield, UK
Department ofHematology, University
of California, Davis, CA
Depart"'!ent ofMedical Microbiology, University
ofLiverpool, Liverpool, UK
Qiagen, Hilden, Germany
Department ofBiology, Massachusetts Institute of Technology, Cambridge,
MA
Departments ofPathology and Neuro-Oncology, The University
of Texas M. D. Anderson Cancer Center, Houston, TX
Department ofPharmacology, University Medical Center
at Stony Brook, Stony Brook, NY