Nonisotopic Probing, Blotting, and Sequencing

By Larry J. Kricka

Since the publication of Nonistopic DNA Probe Techniques in 1992, the move away from radioactive materials for research and diagnostics has continued. This is due in part to public awareness of the hazards of radioactive waste and laws making radioactive disposal more difficult and costly and to improvement in both the sensitivity and convenience of nonisotopic techniques. Several new nonisotopic techniques have been developed and substantial improvements made to existing nonisotopic methods since 1992, and these are now included in Nonisotopic Probing, Blotting, and Sequencing.

Nonisotopic Probing, Blotting, and Sequencing is an updated, expanded edition of the bestseller, Nonisotopic DNA Probe Techniques. It has been thoroughly revised to include the latest improvements in nonisotopic tagging techniques for macromolecules. Like its predecessor, it enables researchers to select the best nonisotopic method for their needs and maximize success by following its straightforward protocols.

• Provides strategies and detailed procedures for labeling, blotting, and probing specific nucleic acid sequences and, with this edition, protein molecules
• Gives protocols for nonisotopic DNA sequencing - new in this edition
• Gives extensive, practical information
• Presents background information for each method
• Provides expert accounts from the inventor or developer of each method
• Contains seven entirely new chapters
• Covers all major types of nonisotopic procedures for labeling and detection

• Language: English
• Publisher: Academic Press
• Release date: Jun 1, 1995
• ISBN: 9780080537665

Book preview

Nonisotopic Probing, Blotting, and Sequencing

Second Edition

Larry J. Kricka

Department of Pathology and Laboratory Medicine, Hospital of the University of Pennsylvania, Philadelphia, Pennsylvania

Academic Press

San Diego   New York   Boston   London   Sydney   Tokyo   Toronto

Table of Contents

Cover image

Title page

Copyright page

Contributors

Preface to the First Edition

Preface to the Second Edition

Part One: Introduction

1: Labels, Labeling, Analytical Strategies, and Applications

I INTRODUCTION

II NUCLEIC ACID HYBRIDIZATION AND BLOTTING

III PROTEIN BLOTTING

IV PATENTS

V CONCLUSIONS

2: Methods for Nonradioactive Labeling of Nucleic Acids

I OVERVIEW

II METHODS FOR ENZYMATIC LABELING

III METHODS FOR CHEMICAL LABELING

IV METHODS FOR CHEMICAL LABELING OF DNA, RNA, AND OLIGODEOXYNUCLEOTIDES WITH MARKER ENZYMES

V OVERVIEW OF FACTORS INFLUENCING HYBRIDIZATION

VI OVERVIEW OF DETECTION SYSTEMS

Part Two: Detection Methods

3: Detection of Alkaline Phosphatase by Time-Resolved Fluorescence

I INTRODUCTION

II MATERIALS

III PROCEDURES

4: Detection of Alkaline Phosphatase by Bioluminescence

I INTRODUCTION

II MATERIALS

III PROCEDURES

IV CONCLUSIONS

5: Detection of DNA on Membranes with Alkaline Phosphatase-Labeled Probes and Chemiluminescent CSPD® Substrate

I INTRODUCTION

II GENERAL SOUTHERN BLOTTING PROCEDURE WITH CHEMILUMINESCENCE

III TWO-STEP HYBRIDIZATION SOUTHERN BLOTTING PROCEDURE—DETECTION OF SINGLE-COPY GENES

IV TROUBLESHOOTING AND COMMENTS

6: Detection of Alkaline Phosphatase by Colorimetry

I INTRODUCTION

II LABELING AND DETECTION STRATEGIES

III HYBRIDIZATION OF BIOTINYLATED PROBES

IV DETECTION OF BIOTINYLATED PROBES

V IN SITU HYBRIDIZATION

VI CONCLUSIONS

ACKNOWLEDGMENTS

7: Detection of Alkaline Phosphatase by Chemiluminescence Using NADP, Ascorbate, and Indolyl Phosphates

I INTRODUCTION

II MATERIALS

III PROCEDURES

IV Discussion

8: Detection of Horseradish Peroxidase by Enhanced Chemiluminescence

I INTRODUCTION

II MATERIALS

III PROCEDURES

9: Detection of Horseradish Peroxidase by Colorimetry

I INTRODUCTION

II MATERIALS

III PROCEDURES

IV CONCLUSIONS

ACKNOWLEDGMENTS

10: Detection of Glucose 6-Phosphate Dehydrogenase by Bioluminescence

I INTRODUCTION

II MATERIALS

III PROCEDURES

IV CONCLUSIONS

11: Detection of Xanthine Oxidase by Enhanced Chemiluminescence

I INTRODUCTION

II MATERIALS

III PROCEDURES

IV CONCLUSIONS

12: Electrochemiluminescent Detection of PCR-Derived Nucleic Acids

I INTRODUCTION

II MATERIALS

III PROCEDURES

IV EXAMPLES

V TROUBLESHOOTING

13: Detection of Phosphors by Phosphorescence

I INTRODUCTION

II MATERIALS

III PROCEDURES

ACKNOWLEDGMENTS

14: Detection of Europium Cryptates by Time-Resolved Fluorescence

I INTRODUCTION

II MATERIALS

III PROCEDURES

15: Detection of Lanthanide Chelates by Time-Resolved Fluorescence

I INTRODUCTION

II INDIRECT LABELING

III CHEMICAL EUROPIUM LABELING OF DNA PROBES

VI ENZYMATIC EUROPIUM LABELING OF DNA PROBES

V EUROPIUM-LABELED OLIGONUCLEOTIDES

VI DETECTION OF MUTATIONS USING LANTHANIDE-LABELED OLIGONUCLEOTIDES

ACKNOWLEDGMENTS

16: Detection of Lanthanide Chelates and Multiple Labeling Strategies Based on Time-Resolved Fluorescence

I INTRODUCTION

II MATERIALS

III PROCEDURES

17: Detection of Acridinium Esters by Chemiluminescence

I INTRODUCTION

II MATERIALS

III PROCEDURES

18: Detection of Energy Transfer and Fluorescence Quenching

I INTRODUCTION

II MATERIALS

III PROCEDURES

Part Three: DNA Sequencing

19: DNA Sequencing by Nonisotopic Methods

I INTRODUCTION

II AUTOMATED FLUORESCENT DETECTION OF DNA SEQUENCES

III MANUAL NONISOTOPIC DETECTION OF DNA SEQUENCES

IV FUTURE DEVELOPMENTS IN NONISOTOPIC DNA SEQUENCING

V CONCLUSIONS

20: Chemiluminescent DNA Sequencing with 1,2-Dioxetanes

I INTRODUCTION

II CHEMILUMINESCENT DNA SEQUENCING PROCEDURE USING BIOTINYLATED PRIMERS

III DETECTION OF BIOTINYLATED DNA SEQUENCING REACTIONS WITH STREPTAVIDIN AND BIOTINYLATED ALKALINE PHOSPHATASE

IV CHEMILUMINESCENT DNA SEQUENCING WITH HAPTENLABELED PRIMERS AND DETECTION WITH ANTIBODY–ALKALINE PHOSPHATASE CONJUGATES

V INSTRUMENTATION

VI TROUBLESHOOTING

VII CONCLUSIONS

Index

Copyright

Front cover photograph: Color enhanced digitized image of a DNA sequence obtained using the chemiluminescent substrate CSPD to visualize bound alkaline phosphatase conjugate. This illustration was kindly provided by Irena Bronstein and Chris Martin of Tropix, Inc.

Copyright © 1995, 1992 by ACADEMIC PRESS, INC.

All Rights Reserved.

No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher.

Academic Press, Inc.

A Division of Harcourt Brace & Company

525 B Street, Suite 1900, San Diego, California 92101-4495

United Kingdom Edition published by

Academic Press Limited

24-28 Oval Road, London NWI 7DX

Library of Congress Cataloging-in-Publication Data

Nonisotopic probing, blotting, and sequencing / edited by Larry J. Kricka

 p. cm.

 Includes index.

 ISBN 0-12-426291-0 (hardcover) 0-12-426292-9(comb bound)

 1. Molecular probes. 2. Immunoblotting. 3. Chemiluminescence assay. 4. Bioluminescence assay. 5. Amino acid sequence. 6. Nucleotide sequence. I. Kricka, Larry J., date.

 QP5 19.9.M65N66  1995

 574.8'8'028--dc20

 94-249 16

 CIP

PRINTED IN THE UNITED STATES OF AMERICA

95   96   97   98   99   00   BC   9   8   7   6   5   4   3   2   1

Contributors

Numbers in parentheses indicate the pages on which the authors’ contributions begin.

Hidetoshi Arakawa (185),     School of Pharmaceutical Sciences, Showa University, Tokyo 142, Hatanodai, Shinagawa-ku, Japan

Lyle J. Arnold, Jr. (391),     Genta, Inc., San Diego, California 92121

Patrick Balaguer (237),     INSERM Unité 58, F-34100 Montpellier, France

Alain Baret (261),     Laboratoire d’Analysis Médicales, Centre de Biologie Médicale, F-44007 Nantes, France

H. Berna Beverioo¹ (285),     Department of Cytochemistry and Cytometry, State University of Leiden, 2300 RA Leiden, The Netherlands

Anne-Marie Boussioux (237),     INSERM Unité 58, F-34100 Montepellier, France

Irena Bronstein(145,493),     TROPIX, Inc., Bedford, Massachusetts 01730

Theodore K. Christopoulos (377),     Division of Clinical Chemistry, Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario, Canada N9B 3P4

Patrik Dahlén (331),     Wallac Oy, FIN-20101 Turku, Finland

Eleftherios P. Diamandis (377),     Department of Clinical Biochemistry, Mount Sinai Hospital, and Department of Clinical Biochemistry, University of Toronto, Toronto, Ontario, Canada M5G 1X5

Ian Durrant (195),     Research and Development, Amersham Laboratories, Amersham International, Amersham, Buckinghamshire HP7911, United Kingdom

Parke K. Flick (475),     United States Biochemical Corporation Research Center, Amersham Life Sciences, Inc., Cleveland, Ohio 44128

Reinhard Erich Geiger (131),     BioAss, D-86911 Giessen, Germany

Eva F. Gudgin Dickson (113),     Department of Chemistry and Chemical Engineering, Royal Military College of Canada, Kingston, Ontario, Canada K7K 5 L0

Pertti Hurskainen (331),     Wallac Oy, FIN-20101 Turku, Finland

Antti Iitia (331),     Department of Biotechnology, University of Turku, FIN-20520 Turku, Finland

Elena D. Katz (271),     Perkin-Elmer Corporation, Norwalk, Connecticut 06859

Christoph Kessler (41),     Biochemical Research Center, Department of Advanced Diagnostics, D-82377 Pensberg, Germany

Larry J. Kricka (3),     Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104

Evelyne Lopez (307),     CIS Bio International, F-30205 Bagnols sur Céze, France

Timo Lövgren (331),     Department of Biotechnology, University of Turku, FIN-20520 Turku, Finland

Masako Maeda (185),     School of Pharmaceutical Sciences, Showa University, Tokyo 142, Hatanodai, Shinagawa-ku, Japan

Chris S. Martin(145,493),     TROPIX, Inc., Bedford, Massachusetts 01730

Géard Mathis (307),     CIS Bio International, F-30205 Bagnols sur Céze, France

Werner Miska (131),     University of Giessen, D-6300 Giessen, Germany

Larry E. Morrison (429),     Vysis, Incorporated, Downers Grove, Illinois 60515

Owen J. Murphy (145),     TROPIX, Inc., Bedford, Massachusetts 01730

Norman C. Nelson (391),     Gen-Probe, Inc., San Diego, California 92121

Quan Nguyen (145),     Genetic Systems Division, Bio-Rad Laboratories, Hercules, California 94547

Jean-Claude Nicolas (237),     INSERM Unité 58, F-34100 Montepellier, France

Alfred Pollak (113),     Allelix Biopharmaceuticals, Mississauga, Ontario, Canada L4V 1 P1

Odette Prat (307),     CIS Bio International, F-30205 Bagnols sur Céze, France

Ayoub Rashtchian (165),     Molecular Biology Research and Development, Life Technologies, Inc., Gaithersburg, Maryland 20877

Mark A. Reynolds (391),     Genta, Inc., San Diego, California 92121

Béatrice Térouanne (237),     INSERM Unité 58, F-34100 Montpellier, France

Akio Tsuji (185),     School of Pharmaceutical Sciences, Shown University, Tokyo 142, Hatanodai, Shinagawa-ku, Japan

Annette Tumolo (145),     Genetic Systems Division, Bio-Rad Laboratories, Hercules, California 94547

Peter C. Verlander² (217),     Department of Investigative Dermatology, Rockefeller University, New York, New York 10021

Marie Agnès Villebrun (237),     INSERM Unité 58, F-34100 Montpellier, France

John C. Voyta (145),     TROPIX, Inc., Bedford, Massachusetts 01730

John M. Wages (271),     Genelabs Technologies, Redwood City, California 94063

Frank Witney (145),     Genetic Systems Division, Bio-Rad Laboratories, Hercules, California 94547

Hector E. Wong (113),     Department of Chemistry, University of Toronto, Toronto, Ontario, Canada M5S 1A1

---

¹Present Address: Department of Cell Biology and Genetics, Erasmus University, 3000 DR, Rotterdam, The Netherlands

²Present Address: ENZO Biochem, Syosset, New York 11791

Preface to the First Edition

Larry J. Kricka

Numerous nonisotopic methods have now been developed as replacements for radioactive labels such as phosphorus-32 and iodine-125 in DNA probe hybridization assays. Most have been developed within the last five years; the range of nonisotopic methods is now so extensive that it is difficult to determine the relative merits and demerits for particular applications.

The objective of this book is to bring together descriptions of the principal nonisotopic methods for DNA hybridization assays, together with experimental details of the methods, including labeling and detection of the label. This book contains descriptions of bioluminescent, chemiluminescent, fluorescent, and time-resolved fluorescent detection methods. It covers the following combinations of label and detection reaction: acridinium esters/chemiluminescence; alkaline phosphatase/bioluminescence, colorimetry, chemiluminescence, time-resolved fluorescence; lanthanide chelates/time-resolved fluorescence; glucose-6-phosphate dehydrogenase/bioluminescence; fluorescence/fluorescence; and horseradish peroxidase/enhanced chemiluminescence/colorimetry. Non-separation DNA probe assay strategies based on selective hydrolysis of acridinium esters and energy transfer involving pairs of probes, one labeled with a chemiluminescent molecule and the other labeled with a fluorophore, are also presented.

Each chapter has been prepared by the inventor or developer of a particular nonisotopic method and thus provides an expert account of the method. Practical details for a range of applications are presented in step-by-step experimental procedures that provide a valuable source of authoritative information.

This book is intended to give research workers and assay developers a single source of information on nonisotopic procedures for DNA hybridization based assays.

Preface to the Second Edition

Larry J. Kricka

Nonisotopic detection methods are an important component of many protein and nucleic acid assay procedures and are slowly replacing the traditional detection methods based on iodine-125 and phosphorus-32. The trend toward nonisotopic methods is being driven by two major factors: the improved sensitivity and analytical convenience (e.g., quick and simple nonseparation assays) of nonisotopic methods and the burdensome regulations that govern the handling and disposal of radioactive materials.

The objective of this book is to provide a unified source of information on the principles and practice of nonisotopic detection methods for protein and nucleic acid blotting, and nucleic acid hybridization and sequencing.

This new edition of Nonisotopic DNA Probe Techniques has been enlarged and expanded to cover both protein and nucleic acid blotting techniques (Western, Southern, and Northern blotting) and DNA sequencing by nonisotopic methods. The chapters have been revised to include new developments in both nucleic acid and protein applications. New chapters have been added on the application of xanthine oxide, ruthenium tris(bipyridyl), phosphor and europium cryptate labels, new chemiluminescent alkaline phosphatase substrates, and DNA sequencing.

Each chapter has been prepared by the inventor or developer of a particular nonisotopic method in order to provide an expert account of the method. Step-by-step protocols and procedures are included in most chapters to facilitate the transfer of these methods into other laboratories.

This new edition is intended as a single and authoritative source of information on nonisotopic methods for students, research workers, and assay developers.

Part One

Introduction

1

Labels, Labeling, Analytical Strategies, and Applications

Larry J. Kricka    Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104

I. Introduction

II. Nucleic Acid Hybridization and Blotting

A. Labels

B. Labeling Procedures

C. Detection of Labels and Nucleic Acid Hybridization Sensitivity

1. Detection Techniques

2. Detection of Nonisotopic Labels

3. Analytical Strategies

III Protein Blotting

IV Patents

V. ConclusionsReference

I INTRODUCTION

Nucleic acid hybridization tests for the detection of specific DNA and RNA sequences and protein blotting assays are now extensively used in research and routine laboratories (Diamandis, 1990; Leary and Ruth, 1989; Matthews and Kricka, 1988; Pollard-Knight, 1991; Rapley and Walker, 1993; Wenham, 1992). These assays have diverse applications in medicine and forensics, and some representative examples of these applications are listed in Table I. Labeled nucleic acid probes are utilized in a variety of assay formats including dot blots, Southern blots (Southern, 1975), Northern blot (Alwine et al., 1977), in situ hybridization, plaque hybridization, and colony hybridization. Western blotting (Burnette, 1981) has become the most prevalent type of protein blotting assay procedure (Table II). An important aspect of these assays is the choice of the substance used to label an assay component and the label detection method. As yet there is no consensus on which substance is the ideal label for detecting either nucleic acids or proteins in the various assay formats. The first assays used a radioactive phosphorus-32 label. However, this label has the major disadvantage of a relatively short half-life (14.2 day) (cf. iodine- 125 used in immunoassay has a half-life of 60 day). Thus nucleic acid hybridization probes have a very short shelf-life. This has placed severe limitations on the routine use and commercialization of probe tests; hence, there are extensive efforts to develop and implement alternatives to the radioactive phosphorus-32 label. Many different substances have been tested as nonisotopic replacements for phosphorus-32, and subsequent chapters of this book provide background and practical details of the application of various nonisotopic labels.

Table I

Applications of Probing and Blotting Assays

Table II

Probing and Blotting Assay Procedures

a Separated on an isoelectric focusing gel.

II NUCLEIC ACID HYBRIDIZATION AND BLOTTING

A Labels

The majority of the substances used as labels for nucleic acids have been tested previously in immunoassay. Nonisotopic labels have been the focus of development because of the limitations of radioactive labels such as phosphorus-32 (Kricka, 1985). These limitations are principally (1) a short half-life that restricts the shelf life of labeled probes and hence hybridization assay kits, (2) possible health hazards during preparation and use of the labeled nucleic acid, and (3) disposal of radioactive waste from the assay. The ideal label for a nucleic acid hybridization probe would have the following properties.

1. Easy to attach to a nucleic acid using a simple and reproducible labeling procedure;

2. Stable under nucleic acid hybridization conditions, typically temperatures up to 80 °C, and exposure to solutions containing detergents and solvents such as formamide;

3. Detectable at very low concentrations using a simple analytical procedure and noncomplex instrumentation;

4. Nonobstructive on the nucleic acid hybridization reaction;

5. Applicable to solution or solid-phase hybridizations. In a solid-phase application, e.g., membrane-based assay, the label must produce a long- lived signal (e.g., enzyme label detected chemiluminescently or by time- resolved fluorescence);

6. Nondestructive. The label must be easy to remove for successive reprobing of membranes. Generally, reprobing is not problematic for phosphorus-32 labels, but it is less straightforward for some nonisotopic labels (e.g., insoluble diformazan product of 5-bromo-4-chloro-3-indolylphosphate (BICP)-nitroblue tetrazolium (NBT)-alkaline phosphatase reaction has to be removed from a membrane with hot formamide);

7. Adaptable to nonseparation (homogenous) formats. Hybridization of labeled DNA probe to its complementary DNA sequence should modulate a property of the label so that it is detectable and distinguishable from unhybridized probe;

8. Stable during storage, providing longer shelf-life for commercial hybridization assay kits; and

9. Compatible with automated analysis. Widespread and large-scale applications of hybridization assays will lead to the need for automated analyzers. The label and the assay for the label must be compatible with a high throughput analyzer (rapid detection using the minimum number of reagents and analytical steps).

None of the labels listed in Table III fulfills all of these criteria and, just as in the case of immunoassays, there is still no agreement on the most appropriate nonisotopic label. Enzymes, such as horseradish peroxidase and alkaline phosphatase, have become particularly popular in recent years as a range of sensitive detection methods has evolved. Alkaline phosphatase, for example, can be detected using chemiluminescent, bioluminescent, and time-resolved fluorescent methods.

Table III

Direct Labels for Nucleic Acid Hybridization Assays

Chemiluminescent compounds

 Acridinium ester

 Isoluminol

 Luminol

Electrochemiluminescent compounds

 Ruthenium /ra(bipyridyl)

 Enzymes

 Alkaline phosphatase

 Bacterial luciferase

 Firefly luciferase

 Glucose oxidase

 Glucose-6-phosphate dehydrogenase

 Hexokinase

 Horseradish peroxidase

 Microperoxidase

 Papain

Renilla luciferase

Enzyme inhibitors

 Phosphonic acid

Fluorescent compounds

 Bimane

 Ethidium

 Europium(III) cryptate

 Fluorescein

 La Jolla Blue

 Methylcoumarin

 Nitrobenzofuran

 Pyrene butyrate

 Rhodamine

 Terbium chelate

 Tetramethylrhodamine

 Texas Red

Metal complexes

 1,4-Diaminoethane platinum complex

Miscellaneous

 Latex particle

 PolyAMP

 Pyrene

Phosphors

 Yttriumoxisulfide (+ europium)

 Zinc silicate (+ arsenic and manganese)

Photoproteins

 Aequorin

Radioluminescent

 Iodine-125

 Phosphorus-32

 Sulfur-35

 Tritium

B Labeling Procedures

Detection of probe : nucleic acid target hybrids can be accomplished by direct or indirect labeling methods. In the former case, a label is attached directly to the nucleic acid by a covalent bond, or the label intercalates noncovalently between the double strand of the probe : nucleic acid target complex. The latter method, indirect labeling, employs a hapten such as biotin (Wilchek and Bayer, 1990) or phosphotyrosine (Misiura et al., 1990) attached to the nucleic acid probe. The hapten is detected using a labeled specific binding protein (e.g., antibiotin, avidin, or streptavidin) (Table IV). A slightly more complex format uses an intermediate binding protein to bridge between the hapten and the labeled binding protein (Table V). Alternatively, a binding protein specific for double-stranded DNA can be used (e.g., monoclonal anti-dsDNA), and complexes are then detected using a labeled antispecies antibody (Mantero et al., 1991). More complex indirect procedures have been developed to improve assay sensitivity (Wilchek and Bayer, 1990). In one design, a biotin-labeled probe is hybridized to the target DNA, followed by reaction of the biotinylated probe with streptavidin. The remaining binding sites on tetravalent streptavidin are then reacted with a biotinylated poly(alkaline phosphatase) to obtain a cluster of alkaline phosphatase labels around the bound biotinylated probe (Leary et al., 1983).

Table IV

Indirect Labels for Nucleic Acid Hybridization Assays

Table V

Indirect Labels for Nucleic Acid Hybridization Assaysa

a That use an intermediate binding protein.

Procedures for the direct labeling of a nucleic acid probe with a hapten or a direct label can be categorized into chemical, enzymatic, and synthetic procedures (Keller and Manak, 1989; Leary and Ruth, 1989; Matthews and Kricka, 1988). One of the goals of a labeling method is to confine the label to sites on the nucleic acid that are not involved in the hydrogen bonding necessary for hybrid formation. However, in practice, hydrogen bonding amino groups are used as labeling sites. This is effective only because the labeling density necessary to provide a detectable probe is low (10–30 modified bases/1000 bases); hence, it does not adversely influenced hybrid stability.

A nucleic acid probe has a limited range of functional groups suitable for attachment of a label; these are listed in Table VI. Various enzymatic labeling procedures have been developed using deoxyribonuclease-DNA polymerase I (Rigby et al., 1977), terminal transferase (Riley et al., 1986), T4 polynucleotide kinase (Maxam and Gilbert, 1977), and the Klenow fragment of Escherichia coli DNA (random hexanucleotide priming reaction) (Feinberg and Vogelstein, 1983). Labeled probes can also be made by cloning the probe sequence into an M13 bacteriophage vector (Hu and Messing, 1982). Polymerase chain reaction (PCR) has also been adapted for labeling, thus allowing simultaneous amplification and labeling (Lion and Haas, 1990; Schowalter and Sommer, 1989). An alternative approach to labeling is to introduce labels or reactive groups for the subsequent attachment of labels during oligomer synthesis (Ruth, 1984; Ruth and Bryan, 1984). Appropriately activated nucleotides can be incorporated either internally (Haralambidis et al., 1987; Ruth et al., 1985) or at the 5′-end (Smith et al., 1985; Sproat et al., 1987). The various types of labeling methods are discussed in detail in Chapter 2.

Table VI

Chemical Labeling of Nucleic Acids

C Detection of Labels and Nucleic Acid Hybridization Sensitivity

The sensitivity of a nucleic acid hybridization assay is determined primarily by the detection limit of the label. This is owing to the sandwich assay design of a hybridization assay and the high binding constant for complementary nucleic acid interaction; thus, low losses due to hybrid dissociation will be encountered. The amount of a specific target DNA sequence in a cell is very small. For example, a mammalian cell contains approximately 6 pg of DNA, comprising 3 billion base pairs or 3 million kB of DNA sequence (a bacterial cell only contains 0.1 pg of DNA). An assay for a typical 1-kB gene using a 1-kB probe requires a detection limit of I attogram of nucleic acid.

Table VII compares the detection limits for various direct and indirect labels. Some of the most sensitive detection schemes involve enzyme labels in combination with either a chemiluminescent or bioluminescent reaction. For example, the detection limit for an alkaline phosphatase label using the adamantyl dioxetane phosphate (AMPPD) is 0.001 attomoles (Bronstein et al., 1990). Results of comparative studies to determine the most effective label-detection reaction combination for a hybridization assay can vary considerably. Urdea et al. (1988) compared the detection limits of seven different labels in a sandwich hybridization assay. The lowest detection limits were obtained using phosphorus-32 and horseradish peroxidase (enhanced chemiluminescent end-point). Another study (Balaguer et al., 1991a) compared luminescent (chemiluminescent or bioluminescent) detection of four different streptavidin–enzyme conjugates. The following signal : blank ratios were obtained after a 5-min incubation in an assay to detect 1 pg of biotinylated pBR 322 spotted on a membrane: glucose-6-phosphate dehydrogenase, 7.0; alkaline phosphatase, 4.5; xanthine oxidase, 2.8; and peroxidase, 1.8. Other investigators have shown different relative detection limits, and more definitive studies are still needed to clarify which is the most effective label and detection reaction for a particular application (e.g., dot blot, in situ hybridization).

Table VII

Detection Limits for Nucleic Acid Labels a

a Detection limits in attomoles. BL, bioluminescence; CL, chemiluminescence; COL, colorimetry; EL, electrochemiluminescence; FL, fluorimetry; RL, radioluminescence. From Arnold et al. (1989); Balaguer et al. (1989); Blackburn et al. (1991); Bronstein and Kricka (1989); Cook and Self (1993); Diamandis (1990); Kricka (1991); Smith et al. (1991a); Urdea et al. (1988).

1 Detection Techniques

The principal types of detection methods for nonisotopic labels are bioluminescence, chemiluminescence, colorimetry, electrochemiluminescence, fluorescence, time-resolved fluorescence and phosphorescence. For some labels several detection methods are possible; these are summarized in Table VIII.

Table VIII

Assays for Selected Nucleic Acid Probe Direct and Indirect Labels

a Chemiluminescence and Bioluminescence

Chemiluminescence is the emission of light that occurs in certain chemical reactions because of decay of chemiexcited molecules to the electronic ground state. Bioluminescence is the chemiluminescence of nature (e.g., the firefly, Photinus pyralis), which involves luciferin substrates and luciferase enzymes, or photoproteins. Both methods are very sensitive (zeptomole amounts, 10− 21 moles), rapid, and versatile [adaptable for hybridizations in solution and on membranes and monitoring with charge coupled device (CCD) cameras] (Hooper and Ansorge, 1990; Wick, 1989).

b Colorimetry

Colorimetric assays produce soluble colored products and are relatively insensitive compared to luminescent assays (chemiluminescence, fluorescence, etc). Most attention has focused on reactions to produce insoluble colored products for locating hybrids on solid phases. These have the advantage of a simple visual read-out and a permanent record, especially for membrane-based assays.

c Electrochemiluminescence

In this process, an electrochemical reaction produces excited-state species that decay to produce a ground state product and light. A disadvantage of this detection reaction is the need for specialized equipment that combines electrochemical-generation and light-detection capabilities. However, an electrochemiluminescence analyzer has now been commercialized (the QPCR System 5000; Perkin-Elmer).

d Fluorescence and Time-Resolved Fluorescence

Fluorescence measurements are capable of detecting single molecules of fluorescein (Mathies and Stryer, 1986). In practice, however, fluorescence is plagued by background signal due to nonspecific fluorescence present in biological samples. Since the background fluoresence tends to be short-lived, it can be avoided by using a long-lived fluorophore (e.g., europium chelate) that is excited by a rapid pulse of excitation light. The fluorescence emission is then measured after the short-lived background has decayed, thus eliminating interference.

e Phosphorescence

Phosphorescence is a long-lived emission from triplet excited states. Inorganic phosphor crystals represent a recent addition to the list of labels for nucleic acids and proteins (Beverloo et al., 1992). Previously, the principal use for this type of compound was in color television screens. The long-lived signal (milliseconds to microseconds) following irradiation with ultraviolet light permits signal acquisition using photographic film or a CCD camera (Seveus et al., 1992).

2 Detection of Nonisotopic Labels

Detection methods for the most popular labels are summarized in the following sections, and further information, together with experimental protocols, is provided in other chapters of this book.

a Acridinium Esters

Oxidation of an acridinium ester by a mixture of sodium hydroxide and hydrogen peroxide produces a rapid flash of light. The detection limit for an acridinium ester-labeled probe is 0.5 amol (Arnold et al., 1989; Weeks et al., 1983). This flash reaction kinetics makes application of acridinium ester labels difficult for membrane-based assays.

b Aequorin

Apoaequorin can now be produced by recombinant DNA techniques (Prasher et al., 1985; Stults et al., 1991). It is converted to aequorin (the photoprotein present in the hydrozoan jellyfish Aequorea) by reaction with coelentrazine, and light emission from aequorin is triggered by calcium ions. Light is emitted as a flash (469 nm); hence, this type of label is difficult to configure in membrane-based applications.

c Alkaline Phosphatase

There is now a series of highly sensitive detection methods for alkaline phosphatase labels. The bioluminescent method uses firefly D-luciferin- O-phosphate as a substrate. An alkaline phosphatase label dephosphorylates the substrate to liberate D-luciferin, which, unlike the phosphate, is a substrate for the bioluminescent firefly luciferase reaction (Hauber et al., 1989; Miska and Geiger, 1987, 1990), see Chapter 4.

Several chemiluminescent assays have also been developed for this enzyme. The most sensitive and widely investigated is based on an adamantyl 1,2-dioxetane aryl phosphate (AMPPD; see Chapter 5). This substance is dephosphorylated to a phenoxide intermediate, which decomposes to form adamantanone and an excited-state aryl ester, which emits light at 477 nm as a protracted glow (> 1 hr) (Bronstein et al., 1990, 1991). Light emission can be enhanced using a variety of compounds including water-soluble polymers {e.g., poly[vinylbenzyl(benzyldimethly ammonium chloride)]} (Bronstein, 1990), and fluoresceinated detergents (e.g., cetyltrimethylammonium bromide) (Schaap et al., 1989). A second generation of dioxetane substrates is now available in which a 5-substituent on the adamantyl ring (e.g., 5-chloro, 5-bromo) prevents aggregation of these molecules (Bronstein et al., 1991), thus reducing reagent background due to thermal degradation.

One of the most popular colorimetric methods for alkaline phosphatase is based on the formation of an insoluble purple dye by sequential dephosphorylation and reduction of BCIP (McGadey, 1970). An alternative substrate is naphthol AS-TR phosphate, which in combination with Fast Red RC, reacts with alkaline phosphatase to produce a red precipitate (Chapter 6).

A salicyl phosphate substrate has been used in a time-resolved assay for alkaline phosphatase. Enzymatic cleavage of the phosphate group produces salicylic acid, which forms a long-lived fluorescent chelate with a europium or terbium ion (Evangelista et al., 1991). Soluble or insoluble chelates can be produced depending on the substituent on the salicylic acid—a fluoro substituent produces a soluble chelate; a branched-chain alkyl group produces an insoluble chelate (Chapter 3).

The proprietary fluorigenic substrate AttoPhos™ (excitation maximum, 440 nm; emission maximum, 550 nm) is another option for detection of alkaline phosphatase. This substrate is superior to methylumbelliferone phosphate and was shown to be 100 times more sensitive in enzyme label detection than a colorimetric substrate in an assay for purified plasmid DNA (pSE9 or pBR322; Cano et al., 1992).

d Europium Chelates

Lanthanides, e.g., europium³ + and terbium³ +, form highly fluorescent chelates with naphthoyltrifluoroacetone or 4,7-te(chlorosulfophenyl)-l, 10-phenanthronline-2,9-dicarboxylic acid (BCPDA) (Diamandis, 1988; Soini and Lovgren, 1987) (see Chapters 10 and 15). These lanthanide chelates have a long-lived fluorescence (100–1000 us) and are useful as time-resolved fluorescent labels. Sensitivity enhancement via multiple labeling can be achieved by coupling the BCPDA to streptavidin and using the multiple-label streptavidin in conjunction with biotinylated probes (Dahlen, 1987; Oser et al., 1990; Syvanen et al., 1986).

e Fluorescein

This fluorophore (fluorescence quantum yield, > 0.85; excitation 492 nm; emission 520 nm) has been used effectively in nonseparation energy transfer probe assays with other fluorophore labels and with chemiluminescent labels (isoluminol) (Morrison et al., 1989) (see Chapter 17).

f Glucose-6-Phosphate Dehydrogenase

A glucose-6-phosphate dehydrogenase label can be measured via the coupled marine bacterial luciferase NAD(P)H: FMN oxidoreductase reaction (Balaguer et al., 1989) (see Chapter 9). (Table VII). The signal, emitted as a glow, is well suited to membrane-based applications.

g Horseradish Peroxidase

The enzyme horseradish peroxidase (HRP) catalyzes the chemiluminescent oxidation of luminol and, in the presence of small amounts of certain phenols (para-iodophenol), naphthols (l-bromo-2-naphthol) and amines (para-anisidine) (enhancers), the analytical features of this reaction are significantly improved (Kricka et al., 1988a; Thorpe and Kricka, 1986). The intensity of the light emission is increased by several orders of magnitude, and background light emission from the luminol-peroxide assay reagent is greatly reduced, which leads to a dramatic increase in the signal: background ratio. The light emission from this reaction is a long-lived glow (> 30 min), suitable for membrane-based assays (Durrant, 1990; Durrant et al., 1990; Matthews et al., 1985; Schneppenheim and Rautenberg, 1987) (see Chapter 8). A dioxetane substrate for HRP has been also described (Urdea and Warner, 1990). HRP cleaves a 2-methylnaphthoxy substituent from the dioxetane to produce AMPPD, which is detected via its alkaline phosphatase-catalyzed chemiluminescent decomposition (vide supra).

Several substrates that produce insoluble colored products are in use, including 3-amino-9-ethylcarbazole (red product), 4-chloro-l-naphthol (blue product), and 3,3′-diaminobenzidine (brown product). 3,3′, 5,5′- Tetramethybenzidine (TMB) is oxidized by HRP to a semisoluble blue cationic dye, which can be trapped by treating the membrane with dextran sulfate (Sheldon et al., 1987).

h Luminol and Isoluminon

Metal ions and peroxidases (e.g., cobalt, microperoxidase) catalyze the chemiluminescent oxidation of luminol and isoluminol. Labeling is most convenient via the aryl amino group, but the chemiluminescence from the luminol label is reduced 10-fold when this amino group is substituted (Schroeder et al., 1978). In contrast, substitution of the aryl amino group of the less efficient isoluminol (chemiluminescence quantum yield: luminol, 0.01, isoluminol, 0.001) causes a 10-fold increase in the quantum yield. Hence isoluminol and its derivatives have been preferred as labels and applied mainly in conjunction with fluorophores in energy-transfer assays (see Chapter 17).

i Phosphors

Protein A can be labeled with stabilized suspensions of a red-emitting yttriumoxisulfide (emission maximum, 620 nm; lifetime, 760 μsec) or a green-emitting zinc silicate phosphor (emission maxium, 522 nm; lifetime, 11 msec). These phosphor protein A conjugates have been tested in Southern and dot blots. Membrane-bound phosphor particles were detected by exposing the membrane to ultraviolet light and detecting the phosphorescent emission on photographic film. In a dot blot assay for lambda DNA, 330 fg was detectable above background (Beverloo et al., 1992). The advantages of the phosphor labels are that the phosphorescent signal is not influenced by the presence of water, pH, or changes in temperature and the range of emission wavelengths permits simultaneous multianalyte assays.

j Renilla Luciferase

Recombinant Renilla (sea pansy) luciferase catalyzes the bioluminescent oxidation of coelenterazine. Light is emitted as a glow (at 480 nm) and, in the presence of green fluorescent protein, energy transfer occurs with a shift in light emission to 508 nm (Ward and Cormier, 1978). This enzyme has been used mainly as a secondary label (biotin conjugate) (Stults et al., 1991).

k Ruthenium Bipyridyl Complexes

Ruthenium and osmium complexes, e.g., ruthenium(II) /m(bipyridyl) t(Ru(bpy)3² +)], can be used as electrochemiluminescent labels and detected by reaction with tripropylamine radicals (Blackburn et al., 1991; Massey et al., 1987). Ru(bpy)² +3 is oxidized at the electrode surface to form Ru(bpy)³ +, which reacts with tripropylamine cation radicals, to produce excited state Ru(bpy)² +3. This species decays to its ground state, and light is emitted at 620 nm.

l Xanthine Oxidase

Xanthine oxidase catalyzes the luminescent oxidation of luminol. The long-lived light emission (lasting for several days) is enhanced by an iron- ethylenediaminetetraacetic acid (EDTA) complex via hydroxyl radical production (Balaguer et al., 1991; Baret et al., 1990).

3 Analytical Strategies

The extreme sensitivity required for the detection of single copy genes has generated several different analytical strategies that attempt to increase specific signal by amplification of target or probe, or to reduce nonspecific background signal by a background rejection technique.

a Amplifying Labels

Amplifying labels initiate cascade reactions, and sensitivity can be further increased by multiple labeling. Those based on the biotin:avidin system are especially effective at increasing detection sensitivity. However, sensitivity can be degraded by background from assay reagents, nonspecific binding of labeled probe, and contaminants.

b Probe Networks

Probe networks provide a further method of introducing an amplification factor into a hybridization assay. This assay utilizes three types of probes (Urdea et al., 1987). A set of short primary probes binds to the target, and these are subsequently reacted with a polymerized second probe, which in turn hybridizes with a multiple enzyme-labeled probe. Amplification of 100-fold can be achieved compared to the signal from a single oligomer probe.

c Probe Amplification

Probes can be amplified using Q-beta replicase. This is an exponential amplification method for RNA probes based on an RNA-directed RNA polymerase (Q-beta replicase) (Table IX). (Kramer et al., 1992: Lizardi et al., 1988). In this method a RNA probe is hybridized to its target, isolated as the hybrid, and then denatured. The released RNA probe is amplified a billion-fold in a single step, and then the vast number of probe copies is quantitated in a secondary hybridization step.

Table IX

Target and Probe Amplification Techniques

a Theoretical amplification = 2n

b Theoretical amplification = (1 + E)n (E, efficiency of the reaction; n, number of cycles)

d Target Amplification

Selective amplification of a DNA target or an RNA target is becoming increasingly popular. A DNA or RNA target can be selectively amplified, thus reducing the requirement for ultrasensitive detection techniques. Several different methods have been developed. The PCR amplifies a DNA target sequence 10 million-fold after 30 cycles (Mullis, 1987; Mullis and Faloona, 1987), and a transcription-based amplification system will produce a 2–5 million-fold amplification of RNA target after 4 cycles (Kwoh et al., 1989). Increasing the amount of analyte reduces the requirement for an ultrasensitive detection technique, but it does add extra steps to the overall analytical procedure. Recent developments have focused on simplification of PCR using either microfabricated devices (Northrup et al., 1993 ; Wilding and Kricka, 1994) or closed vessel automated systems (Findlay, 1993; Findlay et al., 1993).

e Background Rejection

Background rejection makes use of time-resolved fluorescent labels (terbium and europium chelates) (Syvanen et al., 1986) and substrates (e.g., salicyl phosphate–europium chelate) (Evangelista et al., 1991). These end-points utilize long-lived fluorescent chelates activated by a pulse of excitation light. The fluorescent signal is measured after the short-lived background fluorescence has decayed. Interference can also be minimized by appropriate design of the hybridization assay. Thompson et al. (1989) developed a reversible target capture assay, which uses a 3′- poly(dA)-tailed capture probe, a label probe, and an oligo(dT)-labeled paramagnetic particle. The target nucleic acid hybridizes to the probes, and the hybrids are captured via interaction of the dA tail on the capture probe and the dT tail on the paramagnetic particle. The particles are washed, and then the relatively weak bond between the dA and dT tails is disrupted using guanidine thiocyanate. The particles are replaced with fresh particles, and the cycle of capture, washing, and release is repeated. In this way any interferents in solution or bound to the particles are removed before the measurement step.

f Nonseparation Assays

An assay that can be performed in a nonseparation or homogeneous format has several practical advantages. Lengthy, repetitive, and timeconsuming wash steps are avoided; the assay requires only a single incubation step followed by a signal measurement step; and the assay is amenable to automation.

The current range of nonseparation nucleic acid assays is summarized in Table X. The most successful is the hybridization protection assay. This technique exploits the differential hydrolysis of hybridized and nonhybridized acridinium-ester-labeled probes and has been applied to the detection and identification of different infectious organisms (Arnold et al., 1989).

Table X

Nonseparation (Homogeneous) Nucleic Acid Assays

An enzyme-channeling strategy has been developed based on a bioluminescent immunoadsorbent (antifluorescein antibody, marine bacterial luciferase, and NAD(P)H: FMN oxidoreductase co-immobilized on Sepharose beads) (Balaguer et al., 1991). Target DNA is reacted with two probes, one labeled with fluorescein and the other with biotin. The bioluminescent immunoadsorbent captures the complexes via reaction of the immobilized antifluorescein antibody with the fluorescein label on the probe. Biotin label on the captured complexes is then reacted with a streptavidin-glucose-6-phosphate dehydrogenase conjugate. The bioluminescent reaction, initiated by reaction of the glucose 6-phosphate label with a mixture of glucose 6-phosphate and NAD, is more efficient for the bound label (efficient channeling of substrates between the coupled enzymes) than for label in solution. Hence, we can distinguish between bound and unbound label without a separation step.

Energy transfer assay strategies have been devised based on probes labeled with a donor and an acceptor fluorophore (e.g., fluorescein and tetramethylrhodamine) (Cardullo et al., 1988) and on probes labeled with a chemiluminescent compound (isoluminol) and a fluorophore (tetramethylrhodamine) (Heller and Morrison, 1985). Modulation of the steady-state fluorescence polarization of a fluorescein-labeled probe when it binds to a target has also been demonstrated as a viable nonseparation assay strategy (cf., fluorescence polarization immunoassays) (Wang et al., 1992). Devlin et al. (1993) described a transient-state polarized fluorescence assay based on a probe labeled with La Jolla Blue. This assay detected 1 fmol of a 382-base RNA transcript from HIV-1 (generated by a 3SR amplification reaction); the sensitivity of this method was comparable to that of conventional heterogeneous assays using isotopie (³²P) or nonisotopic (e.g., lanthanide chelates) labels.

g Imaging, Multiple Labeling, and Matrix Assays

A growing trend in nucleic acid detection is toward multiple simultaneous assays in two-dimensional formats. Membranes are the traditional solid support for nucleic acid blotting assays. Signal from bound label, such as an alkaline phosphatase label detected with a chemiluminescent substrate, can be conveniently imaged using charge-coupled device (CCD) cameras (Wick, 1989; Hooper and Ansorge, 1990; Hooper et al., 1993; Rushbrooke et al., 1993). This technique is also applicable to tissue sections probed with lanthanide-chelate-labeled probes (Seveus et al., 1992).

A set of probes, each labeled with a different label, provides a means of probing a sample for a range of target sequences. Alkaline phosphatase, glucose-6-phosphate dehydrogenase, horseradish peroxidase, and xanthine oxidase labels were combined in a multiple luminescence procedure for the detection of papillomavirus 11, 16, and 18 in amplified cervical scrapes (Chikhaoui et al., 1992). Combinations of fluorophore labels (Lutz et al., 1992) and combinations of fluorophore labels mixed in different ratios are now widely employed in FISH (fluorescence in situ hybridization) assays in cytogenetics to identify chromosomal abnormalities (Dauwerse et al., 1992; Pinkel, 1993). A combination of 3 labels in different multiple ratios can produce 12 different colors and this permits painting of a chromosome (Morrison, 1993).

An array or matrix of nucleic acid probes immobilized at discrete locations on the surface of a piece of silicon or glass provides a convenient means of simultaneously probing a sample for the presence of different target sequences (Southern et al., 1992; Beattie et al., 1993; Sheldon et al., 1993). Arrays of probes contained in an array of test wells (100 μm²) micromachined in a silicon surface have also been constructed. Hybridization is detected by an electronic device fabricated in each well that measures the change in dielectric relaxation frequency due to the presence of the captured target (Beattie et al., 1993).

III PROTEIN BLOTTING

Protein blotted onto a membrane can be detected with a variety of stains including Coomassie blue, Amido black, and India ink (Harper et al., 1990). These stains have limited sensitivity and have been largely displaced by specific labeled detection agents (labeled antibody, avidin, streptavidin, protein A). Several of the labels listed in Table III have been tested in protein blotting formats such as Western blotting (Towbin et