Clinical Applications of PCR

M E T H O D S I N M O L E C U L A R M E D I C I N E "

John M. Walker, SEIUES EDXrOR

16. Clinical Applications of PCR, edited by E M. Dennis Lo, 1998 15. Molecular Bacteriology: Diagnostic and Experimental Applications,

edited by Nell Woodford and Alan Johnson, 1998 14. Tumor Marker Protocols, edited by Margaret Hanausek and Zbigniew

Walaszek, 1998 13. Molecular Diagnosis of Infectious Diseases, edited by Udo Reischl,

1998 12. Diagnostic Virology Protocols, edited by John R. Stephenson andAlan

Warnes, 1998 11. Therapeutic Application of Ribozymes, edited by Kevin J. Scanlon,

1998 10. Herpes Simplex Virus Protocols, edited by S. Moira Brown

and Alasdair MacLean, 1998 9. Lectin Methods and Protocols, edited by Jonathan M. Rhodes

and Jeremy D. Milton, 1998 8. Helicobacter pylori Protocols, edited by Christopher L. Clayton

and Harry L. T. Mobley, 1997 7. Gene Therapy Protocols, edited by Paul D. Robbins, 1997 6. Molecular Diagnosis of Cancer, edited by Finbarr Cotter, 1996 5. Molecular Diagnosis of Genetic Diseases, edited by

Rob Elles, 1996 4. Vaccine Protocols, edited by Andrew Robinson, Graham H. Farrar,

and Christopher N. Wiblin, 1996 3. Prion Diseases, edited by Harry E Baker and

Rosalind M. Ridley, 1996 2. Human Cell Culture Protocols, edited by Gareth E. Jones, 1996 1. Antisense Therapeutics, edited by Sudhir Agrawal, 1996

Clinical Applications of PCR

Edited by

Y. M. Dennis Lo The Chinese University of Hong Kong, Hong Kong SAR

Humana Press ~ Totowa, New Jersey

© 1998 Humana Press lnc. 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512

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Preface

The polymerase chain reaction (PCR) is one of the most important molecular biological methods ever devised, with numerous applications to clini- cal molecular medicine. Since its description in 1985, PCR has undergone tremendous improvements, and many variations on the basic PCR theme have been published. With such a large volume of PCR-related literature, a clinical scientist wishing to use the technique will have a difficult task locat- ing the relevant information to implement it effectively. There is thus clearly a need for an up-to-date volume with detailed protocols to facilitate the setting up of those techniques most relevant to clinical applications.

Unlike some other books on this topic, Clinical Applications of PCR includes only methods that are of direct relevance in clinical settings. The book is organized in three parts: an introductory section, a section on general methodology, and a final section with specific clinical applications. The first section covers the basic principles of PCR and is most useful to those new to molecular diagnosis. The next chapter includes useful tips for setting up a PCR laboratory. Section 2 then outlines some of the most commonly used PCR-based techniques in molecular diagnosis. Section 3 includes carefully chosen examples that represent typical applications of PCR in diverse clinical fields, encompassing hematology, oncology, genetics, and microbiology.

For clinical applications, two attributes of PCR are especially important: its sensitivity and its ability to detect sequence variations rapidly. Consequently, a significant part of the book is devoted to PCR applications that take advan- tage of these characteristics. The sensitivity of PCR allows single cell analysis and the detection of minority cell/DNA populations to be carried out. Impor- tant clinical applications in those areas covered here include the detection of cir- culating cancer cells in oncology patients and noninvasive prenatal diagnosis using fetal cells in maternal blood. The analysis of sequence variations by PCR is covered extensively in Section 2.

Our hope is that clinical scientists across the many specialties that increasingly require the application of PCR will find Clinical Applications of PCR the highly useful book we have striven to create.

Y. M. Dennis Lo

Contents

Preface ............................................................................................................. v Contributors ..................................................................................................... ix

PART I. INTRODUCTION 1 Introduction to the Polymerase Chain Reaction

Y. M. Dennis Lo ...................................................................................... 3 2 Setting Up a PCR Laboratory

Y. M. Dennis Lo .................................................................................... 11

PART II. GENERAL METHODOLOGY 3 Amplification from Archival Materials

Y. M. Dennis Lo .................................................................................... 21 4 Quantitative PCR

Luc Raeymaekers ................................................................................ 27 5 Mutation Screening Using PCR-SSCP: Silver Staining and Isotopic

Protocols Philip J. Saker ...................................................................................... 39

6 Multiple Fluorescence-Based PCR-SSCP Analysis with Primer-, Post-, and Internal Labeling

Hiroyuki Iwahana and Mitsuo Itakura ............................................... 51 7 The Amplification Refractory Mutation System

Y. M. Dennis Lo .................................................................................... 61 8 Artificial Restriction Fragment Length Polymorphism (A-RFLP)

Analysis Y. M. Dennis Lo and Virginia A. Horton ............................................ 71

9 Long Range PCR William Waggott ................................................................................... 81

10 Generation of Labeled Probes by PCR Y. M. Dennis Lo and Shu F. An .......................................................... 93

11 PCR for the Detection of Minority DNA Populations Y. M. Dennis Lo .................................................................................. 101

12 In Situ Amplification John O' Leary ..................................................................................... 109

vii

vii i Conten ts

13 Sequencing of PCR Products Paul Moss and Swee Lay Thein ....................................................... 145

14 Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) Rajko Kusec ....................................................................................... 149

PART III. CLINICAL APPLICATIONS 15 PCR Amplification of Minisatellite DNA for the Detection

of Mixed Chimerism After Bone Marrow Transplantation Etienne Roux ...................................................................................... 161

Diagnosis of Mitochondrial Disorders Using the PCR Kar l J. Mor ton ..................................................................................... 171

PCR Analysis of CD44 Variants in Tumors John Bolodeoku ................................................................................. 189

Detection of Circulating Solid Tumor Cells by Reverse Transcriptase Polymerase Chain Reaction

Susan A. Burch i l l ............................................................................... 203

Detection of Circulating Tumor Cells Using Immunobead-PCR Jenni fer E. Hard ingham .................................................................... 225

Single Cell PCR: Theory, Practice, and Clinical Applications lan Findlay .......................................................................................... 233

PCR-Based Noninvasive Prenatal Diagnosis Using Fetal Cells in Maternal Circulation

Y. M. Dennis Lo .................................................................................. 265 22 Noninvasive Prenatal Diagnosis Using a Single Fetal Nucleated

Erythrocyte Isolated by Micromanipulation from Maternal Blood Ak ih iko Sekizawa, H i rosh i Saito, Takumi Yanaihara .................... 275

Diagnosis of Chromosomal Aneuploidies Using Quantitative Fluorescent PCR

Barbara Pert l and Matteo Ad ino l f i ................................................... 287

24 Detection of the Hepatitis C Virus by RT-PCR Christopher John Healy and Steven Read ..................................... 301

25 The Application of PCR to the Detection of M. tuberculosis in Sputum Samples

Majella Maher, Maura Glennon, Martin Cormican, and Terry Smith ............................................................................. 309

26 Microsatellite Analysis in Human Disease John Koreth, Christopher J. Bakkenist,

and James O" D. McGee ............................................................... 321 Index ............................................................................................................. 341

16

17

18

19

20

21

23

Contributors

MATTEO ADINOLFI ° Department of Obstetrics and Gynecology, and The Galton Laboratory, University College, London, UK

SHU F. AN • Institute of Neurology, University of London, Queen Square, London, UK

JOHN BOLODEOKU ° Department of Chemical Pathology, School of Medicine, Royal Free Hospital, London, UK

SUSAN A. BURCHILL • Candlelighters Children's Cancer Research Group, Cancer Medicine Research Unit, St. James' University Hospital, Leeds, UK

MARTIN CORMICAN • Department of Immunology, University College Hospital, Galway, Ireland

IAN FINDLAY • Centre Reproduction, Growth, and Development, Institute of Pathology, Algernon Firth Building, University of Leeds, UK

MAURA GLENNON ° Department of Veterinary Science, School of Agriculture and Food Science, Queens University of Belfast, Ireland

J E N N I F E R E . H A R D I N G H A M • Department of Haematology-Oncology, The Queen Elizabeth Hospital, Woodville, S. A., Australia

CHRISTOPHER JOHN HEALEY ° Addenbrooke's Hospital, Cambridge, UK V~RGINIA HORTON • Diabetes Research Laboratories, Radcliffe Infirmary,

University of Oxford, UK MITSUO ITAKURA • Otsuka Department of Clinical and Molecular Nutrition,

University of Tokushima, Japan HIROYUKI IWAHANA • Otsuka Department of Clinical and Molecular

Nutrition, University of Tokushima, Japan JOHN KORETH • Nuffield Department of Pathology and Bacteriology, John

Radcliffe Hospital, University of Oxford, UK RAJKO KUSEC • Voncinina, Zagreb, Croatia Y. M. D~NN]S LO ° Department of Chemical Pathology, Prince of Wales

Hospital, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR

MAJELLA MAHER • The National Diagnostics Centre, University College, Galway, Ireland

KARL J. MORTEN • Neurosciences Group, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK

ix

x Contr ibutors

PAUL M o s s • Institute of Molecular Medicine, John Radcliffe Hospital University of Oxford, UK

JOHN O ' LZARY ° Department of Pathology, Cornell University Medical College, The New York Hospital-Cornell Medical Center, NY

BARBARA PERTL • Department of Obstetrics and Gynecology, University of Graz, Austria

L u c RAEYMAEKERS • Laboratorium Voor Fysiologie Ku Leuven, Campus Gasthuisberg, Leuven, Belgium

STEVEN READ • Department of Virology, PHLS, John Radcliffe Hospital Headington, Oxford, UK

ETIENNE ROUX ° Unitk d'Immunologie de Transplantation, H6pital Cantonal Universitaire de Genkve, Switzerland

HIROSHI SAITO ° Department of Obstetrics and Gynecology, School of Medicine, Showa University, Tokyo, Japan

PmLIP J. SAKER • Unit of Metabolic Medicine, St. Mary's Hospital Medical School Paddington, London, UK

AKIHIKO SEKIZAWA • Department of Obstetrics and Gynecology, School of Medicine, Showa University, Tokyo, Japan

TERRY SMITH ° The National Diagnostics Centre, University College Galway, Ireland

SWEE LAY TH~N ° Institute of Molecular Medicine, John Radcliffe Hospital University of Oxford, UK

WILLIAM WA6GOTT • Department of Cellular Science, John Radcliffe Hospital, University of Oxford, UK

TAKUMI YANAIHARA ° Department of Obstetrics and Gynecology, School of Medicine, Showa University, Tokyo, Japan