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
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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