Advanced Biomedical Technologies

Series Editor Fram;ois Ferre The Immune Response Corporation

Editorial Advisory Board Bruce K. Patterson Northwestern University

Kary B. Mullis Vyrex Inc.

Philip L. Feigner Vical Inc.

William N. Drohan American Red Cross

Michael 1. Heller Nanogen Inc.

Phillips Kuhl Cambridge Healthtech Institute

Michael Karin University of California at San Diego

Robert E. Sobol Sidney Kimmel Cancer Center

Willem P. C. Stemmer Maxygen Inc.

Forthcoming volumes in the series Rational Therapeutic Target in Angiogenesis J.M. Pluda and W.W. Li, editors

Techniques in Localization of Gene Expression Bruce K. Patterson, editor

The Biology of p53 Michael I. Sherman and Jack A. Roth, editors

Molecular Genetic Profiling: Applications to Diagnostics and Disease Management Lance Fors, editor

Gene Quantification

Fran<;ois Ferre Editor

Foreword by Edwin M. Southern

Birkhauser Boston • Basel • Berlin

Fran~is Ferre The Immune Response Corporation Carlsbad, CA 92008, U.S.A.

Library of Congress Cataloging In-Publication Data

Gene quantification I Fran~ois Ferre, editor: foreword by Edwin Southern. p. cm. - (Advanced biomedical technologies) Includes biographical reference and index. ISBN-1 3; 978-1-46 t 2-8682-0 c-ISBN-1 3; 978-1-46 t 2-4164-5 001: 10.1007/978- 1-4612-4164-5 1. Quantitative genetics. 2. Fran~ois. II. Series. QH452.7.G461997 5n.S·6--dc21

Gene amplification. 3. Polymerase chain reaction. I. Ferre,

Printed on acid-free paper © Birkhauser Boston 1998 Birkhauser Copyright is not claimed for works of U.S. Government employees.

97-21838 CIP

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Contents

LIST OF CONTRIBUTORS

FOREWORD Edwin M. Southern

Key Issues, Challenges and Future Opportunities in Gene Quantification Franc;ois Ferre

PART I: METHODSrrECHNOLOGY ISSUES

viii

xi

1

A. GENE QUANTITATION BASED ON PCR AMPLIFICATION

Present and Future Detection Formats for PCR Quantitation of Nucleic Acids 19 Shirley Kwok and John Sninsky

Determination of Target Copy Number of Quantitative Standards Used in PCR Based Diagnostic Assays 31 Zhuang Wang and Joanne Spadoro

Quantification of Specific Nucleic Acids, Regulated RNA Processing and Genomic Polymorphisms Using Reversed-Phase HPLC 45 A. Hayward-Lester, B.S. Chilton, P.A. Underhill, P.J. Oefner, and P.A. Doris

Capillary Electrophoresis for Quantitative Genetic Analysis 79 Jill M. Kolesar and John G. Kuhn

Quantitative PCR Technology 97 Lincoln McBride . .. Mark Oldham

Statistical Estimations of PCR Amplification Rates 111 Jean Peccoud and Christine Jacob

Fluorescence Monitoring of Rapid Cycle PCR for Quantification 129 Carl Wittwer, Kirk Ririe, and Randy Rasmussen

vi Contents

Kinetic Elisa-PCR: A Versatile Quantitative PCR Method 145 Olivier Lantz, Elizabeth Bonney, Scott Umlauf, and Yassine Taoufik

B. GENE QUANTITATION BASED ON OTHER TARGET AMPLIFICATION SYSTEMS

Quantitation of RNA by NASBA TM: Applications and Issues for HIV-1 and AIDS 169 Joseph Romano, Paul van de Wiel, and Stuart Geiger

Application of Transcription-Mediated Amplification to Quantification of Gene Sequences Frank Gonzales and Sherrol H. McDonough

C. GENE QUANTITATION BASED ON SIGNAL AMPLIFICATION

Branched DNA (bDNA) Technology for Direct Quantification of

189

Nucleic Acids: Design and Performance 205 Mark L. Collins, Peter J. Dailey, Lu-Ping Shen, Mickey S. Urdea, Linda J. Wuestehube, and Janice A. Kolberg

Hybrid Capture™ - A Sensitive Signal Amplified Test for the Detection and Quantitation of Human Viral and Bacterial Pathogens 225 Attila T. Lorincz, Mariana G. Meijide, James G. Lazar, and Abel De La Rosa

PART II: APPLICATIONS

Quantification of Gene Expression by Competitive RT-PCR: The hCG{3/LH{3 Gene Cluster 253 Vladimir Lazar, Ivan Bieche, Michel Bahuau, Yres Giovangrandi, Dominique Bellet, and Michel Vidaud

Quantitative Detection of Mycoplasma DNA Using Competitive PCR 265 Maninder K. Sidhu, Mei-June Liao, and Abbas Rashidbaigi

The Detection and Quantification of ber-abl in Chronic Myeloid Leukemia Following Marrow Transplantation 277 Jerald Radich

Contents vii

Competitive RT-PCR Analysis of Brain Gene Expression During Inflammation and Disease 295 Douglas L. Feinstein and Elena Galea

Development and Application of Real-Time Quantitative PCR 313 P. Mickey Williams, Todd Giles, Ayly Tucker, Jane Winer, and Chris Heid

Branched DNA (bDNA) Technology for Direct Quantification of Nucleic Acids: Research and Clinical Applications 327 Janice A. Kolberg .. . Marcia E. Lewis

Quantification of Plasmid DNA Expression in Vivo 343 Marston Manthorpe, Jukka Hartikka, H. Lee Vahlsing, and Michael Sawdey

Index 369

List of Contributors

Michel Bahuau, Laboratoire d'Immunologie des Tumeurs, URA 1484 CNRS et Laboratoire de Genetique MoIeculaire, FacuIte des Sciences Pharmaceutiques et Biologiques de Paris, 4 Avenue de L'Observatoire, 75006 Paris, France Dominique Bellet, Laboratoire d'Immunologie des Tumeurs, URA 1484 CNRS et Laboratoire de Genetique Moleculaire, Faculte des Sciences Pharmaceutiques et Biologiques de Paris, 4 Avenue de L'Observatoire, 75006 Paris, France Ivan Bieche, Laboratoire d'Oncogenetique, Centre Rene Huguenin, 35 Rue Dailly, 92211 Saint Cloud, France Elizabeth Bonney, "The Ghost Lab," NIH, NIAID, LCMI, 4 Center Drive, MSC 0420, Bethesda, MD 20891-0420 Beverly S. Chilton, Department of Cell Biology and Biochemistry, Texas Tech University School of Medicine, 3601 Fourth Street, Lubbock, TX 79430 Mark L. Collins, Nanogen Corporation, 10398 Pacific Center Court, San Diego, CA 92121 Peter. J. Dailey, Chiron Corporation, 4560 Horton Street, Emeryville, CA 94608-2916 Abel De La Rosa, Digene Diagnostics, Inc., 230l-B Broadbirch Drive, SilverSpring,MD 20904 Peter A. Doris, Institute of Molecular Medicine, University of Texas Health Sciences Center, 2121 W. Holcombe Boulevard, Houston, TX 77030 Douglas L. Feinstein, Department of Neurology and Neurochemistry, Division of Neurobiology, Cornell University Medical College, 411 East 69 Street, New York, NY 10021 Francois Ferre, The Immune Response Corporation, 5935 Darwin Court, Carlsbad, CA 92008 Elena Galea, Department of Neurology and Neurochemistry, Division of Neurobiology, Cornell University Medical College, 411 East 69 Street, New York, NY 10021 Stuart Geiger, Advanced Bioscience Laboratories, 5510 Nicholson Lane, Kensington, MD 20895 Todd Giles, Genentech, Inc., 460 Point San Bruno Blvd., South San Francisco, CA 94080-4990

Yves Giovangrandi, Laboratoire d'Immunologie des Tumeurs, URA 1484 CNRS et Laboratoire de Genetique Moleculaire, Faculte des Sciences Pharmaceutiques et Biologiques de Paris, 4 Avenue de L'Observatoire, 75006 Paris, France

Frank Gonzales, Gen-Probe Incorporated, 10210 Genetic Center Drive, San Diego, CA 92121 Jukka Hartikka, Cell Biology, Vical Incorporated, 9373 Towne Centre Drive, San Diego, CA 92121 Amanda Hayward-Lester, Department of Cell Biology and Biochemistry, Texas Tech University School of Medicine, 3601 Fourth Street, Lubbock, TX 79430

Contributors

Chris Heid, Genentech, Inc., 460 Point San Bruno Blvd., South San Fran­cisco, CA 94080-4990 Christine Jacob, INRA, Laboratoire de Biometrie, 78352 Jouy En Josas, France Janice A. Kolberg, Chiron Corporation, 4560 Horton Street, Emeryville, CA 94608-2916 Jill M. Kolesar, School of Pharmacy, University of Wisconsin-Madison, Madison, WI 53706-1515 John G. Kuhn, Department of Pharmacology, The University of Texas Health Science Center San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78284-6220

ix

Shirley Kwok, Roche Molecular Systems, 1145 Atlantic Avenue, Suite 100, Alameda, CA 94501 Olivier Lantz, Service d'Hematologie, Universite Paris-Sud, Le Kremlin­Bicetre 94270 and INSERM unit 25, Hopital Necker, Paris, France Vladimir Lazar, Service de Biologie Oncologique, Institut Gustave Roussy, 39 Rue Camille Desmoulins, 94805 Villejuif Cedex, France James G. Lazar, Digene Diagnostics, Inc., 2301-B Broadbirch Drive, Silver Spring, MD 20904 Marcia E. Lewis, Chiron Corporation, 4560 Horton Street, Emeryville, CA 94608-2916 Mei-June Liao, Interferon Sciences, Inc., 783 Jersey Ave., New Brunswick, NJ 08901-3660 Attila LOrincz, Digene Diagnostics, Inc., 2301-B Broadbirch Drive, Silver Spring, MD 20904 Marston Manthorpe, Cell Biology, Vical Incorporated, 9373 Towne Centre Drive, San Diego, CA 92121 Lincoln McBride, Department of Physiology, Applied Biosystems, Inc., 850 Lincoln Center Drive, Foster City, CA 94404 Sherrol H. McDonough, Gen-Probe Incorporated, 10210 Genetic Center Drive, San Diego, CA 92121 Mariana G Meijide, Digene Diagnostics, Inc., 2301-B Broadbirch Drive, SilverSpring,MD 20904 P. J. Oefner, Department of Biochemistry, Stanford University, Stanford, CA 94305 Mark Oldham, Department of Physiology, Applied Biosystems, Inc., 850 Lincoln Center Drive, Foster City, CA 94404 Jean Peccoud, TIMC-IMAG, Institut Albert Bonniot, Faculte de Medecine de Grenoble, 38706 La Tronche Cedex, France Jerald Radich, Fred Hutchinson Cancer Research Center, 1124 Columbia Street, C2S-023, Seattle, WA 98104-1124 Abbas Rashidbaigi, Interferon Sciences, Inc., 783 Jersey Ave., New Brunswick, NJ 08901-3660 Randy Rasmussen, Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT 84132 Kirk Ririe, Idaho Technology, 149 Chestnut Street, Idaho Falls, ID 83402

x Contributors

Joseph Romano, Advanced Bioscience Laboratories, 5510 Nicholson Lane, Kensington, MD 20895 Michael Sawdey, Cell Biology, Vical Incorporated, 9373 Towne Centre Drive, San Diego, CA 92121 Lu-Ping Shen, Chiron Corporation, 4560 Horton Street, Emeryville, CA 94608-2916

Maninder K. Sidhu, Interferon Sciences, Inc., 783 Jersey Ave., New Brunswick, NJ 08901-3660 John Sninsky, Roche Molecular Systems, 1145 Atlantic Avenue, Suite 100, Alameda, CA 94501, Joanne Spadoro, Roche Diagnostics Systems, Branchburg Township, 1080 US Highway 202 South, Somerville, NJ 08876-3771 Yassine Taoufik, Laboratoire Virus, Neurones et Immunite, Universite Paris-Sud, Le Kremlin-Bicetre 94270 and Service de Microbiologie, Hopital P. Brousse, Villejuif 94807, France

Ayly Tucker, Genentech, Inc., 460 Point San Bruno Blvd., South San Francisco, CA 94080-4990

Scott Umlauf, T Cell Sciences, Inc., Molecular Immunology Department, 119 Fourth Ave., Needham, MA 02194-2725 P. A. Underhill, Department of Biochemistry, Stanford University, Stanford, CA 94305 Mickey S. Urdea, Chiron Corporation, 4560 Horton Street, Emeryville, CA 94608-2916 H. Lee Vahlsing, Cell Biology, Vical Incorporated, 9373 Towne Centre Drive, San Diego, CA 92121 Paul van de Wiel, Organon Teknika International, Zeedijk 58, B-23oo, Turnhout, Belgium Michel Vidaud, Laboratoire d'Immunologie des Tumeurs, URA 1484 CNRS et Laboratoire de Genetique Moleculaire, Faculte des Sciences Pharmaceutiques et Biologiques de Paris, 4 Avenue de L'Observatoire, 75006 Paris, France

Zhuang Wang, Roche Diagnostics Systems, Branchburg Township, 1080 US Highway 202 South, Somerville, NJ 08876-3771 P. Mickey Williams, Genentech, Inc., 460 Point San Bruno Blvd., South San Francisco, CA 94080-4990 Jane Winer, Genentech, Inc., 460 Point San Bruno Blvd., South San Fran­cisco, CA 94080-4990 Carl Wittwer, Department of Pathology, University of Utah School of Medicine, 50 North Medical Drive, Salt Lake City, UT 84132 Linda J. Wuestehube, Chiron Corporation, 4560 Horton Street, Emeryville, CA 94608-2916

Foreword

Geneticists and molecular biologists have been interested in quantifying genes and their products for many years and for various reasons (Bishop, 1974). Early molecular methods were based on molecular hybridization, and were devised shortly after Marmur and Doty (1961) first showed that denaturation of the double helix could be reversed - that the process of molecular reassociation was exquisitely sequence dependent. Gillespie and Spiegelman (1965) developed a way of using the method to titrate the number of copies of a probe within a target sequence in which the target sequence was fixed to a membrane support prior to hybridization with the probe - typically a RNA. Thus, this was a precursor to many of the methods still in use, and indeed under development, today. Early examples of the application of these methods included the measurement of the copy numbers in gene families such as the ribosomal genes and the immunoglo­bulin family. Amplification of genes in tumors and in response to drug treatment was discovered by this method.

In the same period, methods were invented for estimating gene num­bers based on the kinetics of the reassociation process - the so-called Cot analysis. This method, which exploits the dependence of the rate of reassociation on the concentration of the two strands, revealed the presence of repeated sequences in the DNA of higher eukaryotes (Britten and Kohne, 1968). An adaptation to RNA, Rot analysis (Melli and Bishop, 1969), was used to measure the abundance of RNAs in a mixed population. A major result of this work was the discovery that the abundance of differ­ent mRNAs in any cell type can vary over several orders of magnitude (Hastie and Bishop, 1976). This observation has implications for methods now in use or under development which are based on the analysis of cDNA clone collections made from whole cell popUlations; without normal­ization, such collections are likely to contain a few sequences represented many times and a large number of sequences present in low numbers or absent.

Gel transfer methods, though designed for qualitative analysis, (South­ern, 1975) were used as a means to measure and compare gene numbers. Related methods for RNA (Alwine, Kemp and Stark, 1977) were applied to the quantitative analysis of gene expression. Methods for applying multiple targets on a single membrane - the dot blot (Kafatos, Jones and Efstratiadis, 1979) - expanded the usefulness of molecular hybridization and with the advent of cloning, this approach could be used to analyze expression levels of individual genes. However, these gene transfer tech-

xii Foreword

niques required millions of copies of a given gene for detection and quantitation.

The advent of amplification methods, such as PCR (Mullis and Faloona, 1987), allowed gene quantification to be performed from very small amounts of starting copy numbers. The focus of this book is to describe how amplification techniques can be harnessed to become quanti­tative methods. These methods have been very successful for individual gene quantification. A case in point is the use of a variety of quantitative methods, described in this book, to measure HIV -1 viral load in HIV-1 infected individuals.

As for all measurements, accuracy and sensitivity are crucial issues, although some applications are more demanding than others; detecting a gene deletion may require sensitivity that will detect a single copy within a whole genome with an accuracy that can distinguish one from two copies. On the other hand, estimating the level of a gene's expression may require only that a high level of expression can be distinguished from a level close to zero.

Recent interest in genome-wide analysis have prompted the develop­ment of methods capable of detecting hundreds of genes or gene products simultaneously. These methods capitalized on the development of robotic spotting devices (Nizetic et at, 1991) that initially applied samples to per­meable membranes. More recently, to achieve higher density, probes (cDNA or oligonucleotides) have been spotted on glass supports (Schena et at, 1995; Lockhart et at, 1996). Current effort is directed at rendering these methods quantitative. The potential to address biological problems by genetic analysis on a genome-wide scale may revolutionize the life sciences. The engine that is driving the revolution is the set of massively parallel procedures made possible by merging molecular biology with tech­niques adopted from the semiconductor industry. Developments that will lead to mUltigene quantitation are principally due to reduction in scale achieved through microfabrication, and to the automation of production methods, of sample preparation, and handling of data production and analysis.

In all the excitement generated by this explosive growth in gene quan­tification methodology, it is important to reflect on questions defined for us by those with a profound understanding of biological systems. It is impor­tant, too, to recognize that many of these new methods have a basis in old ones, and that some of the basic problems we are trying to solve may already have been dealt with. The reader should be aware that some classic papers on gene quantification have publication dates earlier than the ab­stracting services.

- Edwin M. Southern

Foreword xiii

Alwine JC; Kemp DJ; Stark GR (1977) Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl paper and hy­bridization with DNA probes. Proc Natl Acad Sci USA 1977 Dec; 74(12):5350--4

Bishop JO (1974) The gene numbers game. Cell 1974 Jun; 2(2):81--6

Britten RJ; Kohne DE (1968) Repeated sequences in DNA. Hundreds of thousands of copies of DNA sequences have been incorporated into the genomes of higher organisms. Science 1968 Aug 9; 161(841):529-40

Gillespie D; Spiegelman S (1965) A quantitative assay for DNA-RNA hybrids with DNA immobilized on a membrane. J Mol BioI 1965 Jul; 12(3):829-42

Hastie ND; Bishop JO (1976) The expression of three abundance classes of messenger RNA in mouse tissues. Cell 1976 Dec; 9(4 PT 2):761-74

Kafatos FC; Jones CW; Efstratiadis A (1979) Determination of nucleic acid sequence homologies and relative concentrations by a dot hybridization procedure. Nucleic Acids Res 1979 Nov 24; 7(6):1541-52

Lockhart DJ; Dong H; Byrne MC; Folletie MT; Gallo MV; Chee MS; Mittman M; Wang C; Kobayashi M; Horton H; Brown E (1996) Expression monitoring by hybridisation to high-density oligonucleotide arrays. Nature Biotechnol1996 Dec 14; 14:1675-83.

Marmur J; Doty P (1961) Thermal renaturation of deoxyribonucleic acids. J Mol BioI 1961; 3:585-94

Melli M; Bishop JO (1969) Hybridization between rat liver DNA and complementary RNA. J Mol Bioi 1969 Feb 28; 40(1):117-36

Mullis KB; Faloona FA (1987) Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Meth Enzymol1987; 1455:335-50

Nizetic D; Zehetner G; Monaco AP; Gellen L; Young BD; Lehrach H (1991) Construction, arraying, and high-density screening of large insect libraries of human chromosomes X and 21: their potential use as reference libraries. Proc Natl Acad Sci USA 1991 Apr 15; 88(8):3233-7

xiv Foreword

Schena M; Shalon D; Davis RW; Brown PO (1995) Quantitative monitor­ing of gene expression patterns with a complementary DNA microarray. Science 1995 Oct 20; 270(5235):467-70

Southern EM (1975) Detection of specific sequences among DNA frag­ments separated by gel electrophoresis. J Mol Bioi 1975 Nov 5; 98(3):503-17