Protocols for Gene Analysis

Methods in Molecular Biology John M. Walker, SERIES EDITOR

31. Protocols for Gene Analysis, edited by Adrian J. Harwood, 1994 30. DNA-Prote in Interact ions, edited by G. GeoffKneale, 1994 29. Chromosome Analysis Protocols, edited by John R. Gosden, 1994 28. Protocols for Nucleic Acid Analysis by Nonradioact ive Probes, edited

by Peter G. Isaac, 1994 27. B iomembrane Protocols: II. Architecture and Function, edited by

John M. Graham and Joan ,~ Higgins, 1994 26. Protocols for Oligonucleotide Conjugates, edited by Sudhir Agrawa~ 1994 25. Computer Analysis of Sequence Data: Part II, edited by

Annette M. Griffin and Hugh G. Griffin, 1994 24. Computer Analysis of Sequence Data: Part I, edited by

Annette M. Griffin and Hugh G. Griffin, 1994 23. DNA Sequencing Protocols, edited byHugh G. Griffin

and Annette M. Griffin, 1993 22. Optical Spectroscopy, Microscopy, and Macroscopic Technique~

edited by Christopher Jones, Barbara Mulloy, and Adrian H. Thomas, 1994

21. Protocols in Molecular Parasitology, edited by John K Hyde, 1993 20. Protocols for Oligonucleotides and Analogs~ edited by

Sudhir Agrawa~ 1993 19. B iomembrane Protocols: I. Isolation and Analysis, edited by

John M. Graham and Joan A. Higgins, 1993 18. Transgenesis Techniques, edited by David Murphy

and David A. Carter, 1993 17. Spectroscopic Methods and Analyses, edited by Christopher Jones,

Barbara Mulloy, and Adrian H. Thomas, 1993 16. Enzymes of Molecular Biology, edited by Michael M. Burrell, 1993 15. PCR Protocols, edited by Bruce ,4. White, 1993 14. Glycoprotein Analysis in Biomedicine, edited by Elizabeth F. Hounsel~ 1993 13. Protocols in Molecular Neurobiology, edited by Alan Longstaf f

and Patricia Revest, 1992 12. Pulsed-Field Gel Electrophoresis , edited by Margit Burmeister

and Levy Ulanovsky, 1992 11. Prac t ica l Pro te in Chromatography, edited by Andrew Kenney

and Susan Fowell, 1992 10. Immunochemica l Protocols , edited by Margaret M. Manson, 1992 9. Pro tocols in Human Molecular Genetics, edited by

Christopher G. Mathew, 1991 8. Prac t ica l Molecular Virology, edited by Mary IL L. Collins, 1991 7. Gene Transfer and Expression Protocols, edited by Edward J. Murray, 1991 6. P lan t Cell and Tissue Culture, edited by Jeffrey W. Pollard

and John M. Walker, 1990 5. Animal Cell Culture, edited by Jeffrey W. Pollard and John M. Walker, 1990

Protocols for Gene Analys is

Edited by

A d r i a n J. H a r w o o d Medical Research Council, Cambridge, UK

Humana Press ~ Totowa, New Jersey

© 1994 Humana Press Inc. 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512

All rights reserved.

No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise without written permission from the Publisher.

Photocopy Authorization Policy: Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients, is granted by Humana Press, provided that the base fee of US $3.00 per copy, plus US $00.20 per page, is paid directly to the Copyright Clearance Center at 222 Rosewood Dr., Danvers, MA 01923. For those organizations that have been granted a photocopy license from the CCC, a separate system of payment has been arranged and is acceptable to Humana Press. The fee code for users of the Transactional Reporting Service is: [0-89603-258-2/94 $3.00 + $00.20].

Printed in the United States of America. I0 9 8 7 6 5 4 3 2

Library of Congress Cataloging in Publication Data

Main entry under title:

Methods in molecular biology.

Protocols for gene analysis / edited by Adrian J. Harwood. p. cm.---(Methods in molecular biology; 31)

Includes bibliographical references and index. ISBN 0-89603-258-2 I. Gene mapping. 2. Nucleotide sequence. I. Harwood, Adrian J.

II. Series: Methods in molecular biology (Totowa, N.J.); 31 QH445.2.P76 1994 574.87'322---dc20 94-2365

CIP

Preface

It is now twenty years since Cohen and Boyer's first steps into DNA cloning. In the time since then, there has been an ever increasing accel- eration in the development and application of the cloning methodology. With the recent development of the polymerase chain reaction, a second generation of the technology has been born, enabling the isolation of DNA (and in particular, genes) with little more information than the par- tial knowledge of the sequence. In fact, DNA sequencing is now so advanced that it can almost be carried out on the industrial scale. As a consequence of these advances, it now appears feasible to sequence whole genomes, including one the size of the human. What are we going to do with this information? The future of basic molecular biology must lie in the ability to analyze DNA (and especially the genes within it) starting at the DNA level. It is for these problems that Protocols for Gene Analysis attempts to offer solutions.

So you have a piece of DNA, possibly a gene--what do you do next? The first section of this book contains a number of "basic" tech- niques that are required for further manipulation of the DNA. This sec- tion is not intended to be a comprehensive collection of methods, but merely to serve as an up-to-date set of techniques. I refer you to other volumes in the Methods in Molecular Biology series for further recom- binant DNA techniques.

The rest of the volume is broken up into discrete sections that should be perused according to one's individual research interests. Part 2 details a number of methods for in vitro mutagenesis--a direct means of inves- tigating function, whether it is the control of gene expression or the func- tion of the gene product. Part 3 describes a number of methods and electrophoretic techniques for the elucidation of genomic structure. Part 4 offers an excursion into an area that is becoming more important as the number of those genes involved in inherited diseases increases. To study

vi Preface

the polymorphic nature of the genetic pool within a population, it is nec- essary to be able rapidly to detect the sequence variation within it. A number of technical innovations are described that make this possible. Part 5 returns to the more general area of gene expression, and includes a number of techniques vital to the understanding of gene transcription.

The final two sections take the reader far onto another molecular biology frontier. Both ultimately concern the interaction between differ- ent genetic elements, and how it is now possible to step from one to another. Part 6 describes the identification of elements within DNA sequences that bind to proteins, and how this information can be used to isolate the genes encoded by these proteins. In a similar manner, Part 7 describes methods for protein production from cloned DNA and the iden- tification of novel proteins through their association with that DNA. It also takes the process one step farther and describes how ligand binding alone can be used to isolate proteins of interest. At this point, the analy- sis has returned to square one.

Protocols for Gene Analysis has been prepared in the same manner as the previous volumes of the Methods in Molecular Biology series. Each chapter is written in a recipe-like format and designed for direct practical use within the laboratory. Each chapter therefore stresses the practical details, is readily replicable, describes variant versions of the technique, and discusses the problems (and solutions) encountered by their expert authors.

Finally, I would like to thank my colleagues for helpful suggestions and the diligent contributors for all their work and patience through the editorial process.

Adrian J. Harwood

Contents

Preface ....................................................................................................................... v

Contributors .............................................................................................................. xi

PART I. BASIC RECOMBINANT DNA TECHNIQUES

CH. 1. Transformation of Bacteria by Electroporation, Lucy Drury ......................................................................................... 1

CH. 2. Direct Cloning of ~,gtl 1 cDNA Inserts Into a Plasmid Vector, Matthew L. Poulin and lng-Ming Chiu ............................................ 9

CH. 3. PCR Cloning Using T-Vectors, Michael K. Trower and Greg S. Elgar ............................................ 19

CH. 4. Thermal Cycle Dideoxy DNA Sequencing, Barton E. Slatko ............................................................................... 35

PART II. IN VITRO MUTAGENESIS

CH. 5. Ordered Deletions Using Exonuclease III, Denise Clark and Steven Henikof f .................................................. 47

CH. 6. Site-Directed Mutagenesis Using a Double-Stranded DNA Template, Stdphane ViviUe ............................................................................... 57

CH. 7. Site-Directed Mutagenesis Using a Uracil-Containing Phagemid Template,

Michael K. Trower ........................................................................... 67 CH. 8. Construction of Linker-Scanning Mutations by Oligonucleotide Ligation,

Grace M. Hobson, Patricia P. Harlow, and Pamela A. Benfield... 79 CH. 9. Construction of Linker Scanning Mutations Using the Polymerase Chain

Reaction, Patricia P. Harlow, Grace M. Hobson, and Pamela A. Benfield... 87

CH. 10. Localized Random Polymerase Chain Reaction Mutagenesis, Claude G. Lerner and Masayori Inouye ......................................... 97

PART III. GENOMIC STRUCTURE

CH. 11. The Simultaneous Isolation of RNA and DNA from Tissues and Cultured Cells,

Frank Merante, Sandeep Raha, Juta K. Reed, and Gerald Proteau .............................................................. 113

vii

viii Contents

CH. 12. Physical Mapping of the Human Genome by Pulsed Field Gel Electrophoresis,

Jaequeline Boultwood .................................................................... 121 CH. 13. Field Inversion Gel Electrophoresis,

Christoph Heller ............................................................................. 135 Ca. 14. Enhanced Chemiluminescent Detection of Horseradish Peroxidase Labeled

Probes, fan Durrant .................................................................................... 147

CH. 15. Nonradioactive Oligonucleotide Probe Labeling, fan Durrant and Sue Fowler ......................................................... 163

Ca. 16. Analysis of DNA Restriction Enzyme Digests by Two-Dimensional Electrophoresis in Agarose Gels,

Edward 1., Kuf f and Judy A. Mietz ................................................ 177 CH. 17. Inverse Polymerase Chain Reaction,

Daniel h Hartl and Howard Ochman .......................................... 187

PART IV. SEQUENCE VARIATIONS

CH. 18. Use of Silver Staining to Detect Nucleic Acids, Lloyd G. Mitchell, Angelika Bodenteich, and Carl R. Merril ...... 197

CH. 19. A Nonradioactive Method for the Detection of Single-Strand Conformational Polymorphisms (SSCP),

Peter J. Ainsworth and David I. Rodenhiser ................................ 205 CH. 20. Temperature Gradient Gel Electrophoresis (TGGE) for the Detection

of Polymorphic DNA and RNA, Karsten Henco, Jutta Harders, Ulrich Wiese,

and Detlev Riesner ................................................................ 211 CH. 21. TGGE in Quantitative PCR of DNA and RNA,

Jie Kang, Jutta Harriers, Detlev Riesner, and Karsten Henco ..... 229 Ca. 22. The PGK-PCR Clonality Assay (PPCA),

Lambert Busque and D. Gary Gilliland ........................................ 237 CH. 23. Direct Sequencing of PCR Products,

Geraldine A. Phear and Janet Harwood ....................................... 247

PART V. GENE EXPRESSION

CH. 24. The Use of Riboprobes for the Analysis of Gene Expression, Dominique Belin ............................................................................ 257

CH. 25. Quantification of Absolute Amounts of Cellular Messenger RNA by RNA-Excess Solution Hybridization,

Rai Ajit K. Srivastava and Gustav Schonfeld ................................ 273 Measurements of Rate of Transcription in Isolated Nuclei by Nuclear

"Run-Off' Assay, Rai Ajit K. Srivastava and Gustav Schonfeld ................................ 281

CH. 26.

Con~n~

CH. 27. An RNA Polymerase II In Vitro Transcription System, Roberto Mantovani ........................................................................ 289

CH. 28. S1 Mapping Using Single-Stranded DNA Probes, Stdphane ViviUe and Roberto Mantovani ..................................... 299

Ca. 29. Single Primer-Mediated Polymerase Chain Reaction, Rend L, Myers and Ing-Ming Chiu ............................................... 307

PART VI. PROTEIN-DNA INTERACTIONS

CH. 30. In Vivo DNA Footprinting by Linear Amplification, Hans Peter Saluz and Jean-Pierre Jost ........................................ 317

CH. 31. DNA Photofootprinting with Rh(phi)2bpy~+, Scott L, Klakamp and Jacqueline K. Barton ................................. 331

CH. 32. The Gel Retardation Assay, Valerie Scott, Andrew R. Clark, and Kevin Docherty ................... 339

CH. 33. The Southwestern Assay, Jacques Philippe ............................................................................ 349

CH. 34. Cloning DNA Binding Proteins from eDNA Expression Libraries Using Oligonucleotide Binding Site Probes,

lan G. CoweU and Helen C. Hurst ................................................ 363

PART VII. PROTEIN FUNCTION

CH. 35. 6xHis-Ni-NTA Chromatography as a Superior Technique in Recombinant Protein Expression/Purification,

Joanne Crowe, Heinz Diibeli, Reiner Gentz, Erich Hochuli, Dietrich Stiiber, and Karsten Henco .................................... 371

CH. 36. Production of 35S-Labeled Proteins in E. coli and Their Use as Molecular Probes,

Michael A. Lydan and Danton H. O'Day ..................................... 389 CH. 37. Preparation and Ligand Screening of a ~,gtl 1 Lysogen Library,

Rupert Mutzel ................................................................................. 397 Index ...................................................................................................................... 409

Contributors

PETER J. AINSWORTH ° Child Health Research Institute and Department of Pediatrics, Children's Hospital of Western Ontario and University of Western Ontario, London, Ontario, Canada

JACQUELINE K. BARTON ° Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA

DOMINIQUE BELIN ° Department of Pathology, University of Geneva Medical School CMU, Geneva, Switzerland

PAMELA A. BEr~ELO ° The DuPont Merck Pharmaceutical Company, Experimental Station, Wilmington, DE

ANGELIKA BODENTEICH • Laboratory of Biochemical Genetics, NIMH Neuroscience Center at St. Elizabeth's Hospital, Washington, DC

JACQUELINE BOULTWOOD ° Molecular and Cytogenetic Haematology Unit, Department of Haematology, John Radcliffe Hospital, Headington, Oxford, UK

LAMBERT BUSQUE ° Division of Hematology and Oncology, Harvard Medical School, Brigham and Women's Hospital, Boston, MA

IN6-MING CHIU ° Departments of Molecular Genetics and Internal Medicine, The Ohio State University, Davis Medical Research Center, Columbus, OH

ANDREW R. CLARK ° Department of Medicine, University of Birmingham, Queen Elizabeth Hospital, Birmingham, UK

DENISE CLARK ° Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, WA

IAN G. COWELL • Imperial Cancer Research Fund, Gene Transcription Laboratory, Hammersmith Hospital, Du Cane, London, UK

JOANNE CROWE • QIAGEN Inc., Chatsworth, CA HEINZ DOBELI ° F. Hoffman-La Roche Ltd., Pharmaceutical Research-

New Technologies, Basel, Switzerland

xi

xii Contributors

KEVIN DOCHERTY ° Department of Medicine, University of Birmingham, Queen Elizabeth Hospital, Birmingham, UK

LucY DRURY • Imperial Cancer Research Fund, Clare Hail Laboratories, South Mimms, Herts, UK

IAN DtmRAWr • Research and Development, Amersham International Amersham, Bucks, UK

GREC, S. ELC, AR • Molecular Genetics Unit, MRC Centre, Cambridge, UK SUE FOWLER • Research and Development, Amersham International,

Amersham, Bucks, UK REINER GErcrz • F. Hoffmann-La Roche Ltd., Pharmaceutical

Research New Technologies, Basel, Switzerland D. GARY GILLILAND • Division of Hematology and Oncology, Harvard

Medical School, Brigham and Women's Hospital, Boston, MA PATPaCIA P. HAP.LOW • The DuPont Merck Pharmaceutical Company,

Experimental Station, Wilmington, DE JUTrA HARDERS • DIAGEN GmbH, Hilden, Germany DANIEL L. HARTL • Department of Genetics, Washington University

School of Medicine, St. Louis, 11/10 JANET HARWOOD ° Imperial Cancer Research Fund, Clare Hail

Laboratories, South Mimms, Herts, UK CHRISTOPH HEELER • Laboratoire de Physico Chimie Th~orique, Ecole

Superieur de Physique et de Chimie IndustrieUes, Paris, France KARSTEN HENCO • DIA GEN GmbH, Hilden, Germany STEVEN HEr, aKOFF • Howard Hughes Medical Institute, Fred

Hutchinson Cancer Research Center, Seattle, WA ERICH HOCHtrLI ° F. Hoffman-La Roche Ltd., Pharmaceutical

Research-New Technologies, Basel, Switzerland HELEN C. HURST • Imperial Cancer Research Fund, Gene

Transcription Laboratory, Hammersmith Hospital, Du Cane, London, UK

GRACE M. HOBSON ° Alfred L DuPont Institute, Wilmington, DE MASAYOm INOUYE • Department of Biochemistry, University

of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School at Rutgers, Piscataway, NJ

JEAN-PmRRE JOST • FMI, Basel, Switzerland

Contributors xiii

JIE KANG • DIAGEN GmbH, Hilden, Germany SCOTT L. KLAKAMP" Division of Chemistry and Chemical Engineering,

California Institute of Technology, Pasadena, CA EDWARD L. KUFF • Laboratory of Biochemistry, National Cancer

Institute, National Institutes of Health, Bethesda, MD CLAUDE G . LERNER • Abott Laboratories, Anti-Infective Research

Division of Pharmaceutical Discovery, Abott Park, IL MICHAEL A. LYDAN • Department of Zoology, Erindale College,

University of Toronto, Mississauga, Ontario, Canada ROBERTO MANTOVANI • DOI/LGME, Facultd de Mddecine, Strasbourg

Cedex, France FRANK MERArCrE , Department of Genetics, Research Institute,

The Hospital for Sick Children, Toronto, Ontario, Canada CARL R. MERRm • Laboratory of Biochemical Genetics, NIMH

Neuroscience Center at St. Elizabeth's Hospital, Washington, DC JUDY A. MmTz ° Laboratory of Biochemistry, National Cancer

Institute, National Institutes of Health, Bethesda, MD LLOYD G . MITCHELL • Laboratory of Biochemical Genetics, N1MH

Neuroscience Center at St. Elizabeth's Hospital, Washington, DC RUPERT MUTZEL • Fakultiit fiir Biologie, Universit~it Konstanz,

Konstanz, Germany RENI~ L. MYERS • Departments of Molecular Genetics and Internal

Medicine, The Ohio State University, Davis Medical Research Center, Columbus, OH

DANTON n . O ' D A Y • Department of Zoology, Erindale College, University of Toronto, Mississauga, Ontario, Canada

HOWARD OCrtMAN • Department of Genetics, Washington University School of Medicine, St. Louis, MO

GERALDINE A. PHEAR ° Department of Radiology, Health Sciences Center, University of Utah, Salt Lake City, UT

JACQUES PHILIPPE ° Department of Genetics and Microbiology, Centre Medical Universitaire, Geneva, Switzerland

MATTI4EW L. POULIN ° Departments of Molecular Genetics and Internal Medicine, The Ohio State University, Davis Medical Research Center, Columbus, OH

xiv Contributors

GERALD PROTEAU • Department of Zoology, Erindale College, University of Toronto, Mississauga, Ontario, Canada

SANDEEP RAHA • Department of Biochemistry, Erindale College, University of Toronto, Mississauga, Ontario, Canada

JUTA K. REED • Departments of Biochemistry and Chemistry, Erindale College, University of Toronto, Mississauga, Ontario, Canada

DETLEV RIESNER • lnstitut fiir Physikalische Biologie, Heinrich-Heine- Universitiit, Diisseldo~ Germany

DAVID I. RODENHISER • Child Health Research Institute and Department of Pediatrics, Children's Hospital of Western Ontario and University of Western Ontario, London, Ontario, Canada

HANS PETER SALUZ • HKI, Jena, Germany GUSTAV SCHONFELD • Department of Internal Medicine, Washington

University School of Medicine, St. Louis, MO VALERIE S C O T t • Department of Medicine, University

of Birmingham, Queen Elizabeth Hospital, Birmingham, UK BARTON E. SLATKO • New England Biolabs, Inc., Beverly, MA R A I A J I T K , SRIVASTAVA • Department of Internal Medicine,

Washington University School of Medicine, St. Louis, MO DETRtCH STOBER • F. Hoffman-La Roche Ltd., Pharmaceutical

Research-New Technologies, Basel, Switzerland MICHAEL K. TROWER • Molecular Genetics Unit, MRC Centre,

Cambridge, UK. Present Address: Biochemical Targets Department, Microbiology Division, Glaxo Group Research Ltd., Greenford, Middlesex, UK

STI~PHANE VIVILLE • Wellcome Trust Institute of Cancer and Developmental Biology, Cambridge, UK

ULPaCH WmSE • Institut fiir Physikalische Biologie, Heinrich-Heine- Universitiit, Diisseldo~ Germany