Table Of ContentSPRINGER
LAB MANUALS
Springer-V erlag Berlin Heidelberg GmbH
Christoph Kessler (Ed.)
Nonradioactive Analysis
of Biomolecules
Second Edition
With 105 Figures, Nine in Color
Springer
PRIV.-DOZ. DR. CHRISTOPH KESSLER
Roche Diagnostics GmbH
Roche Molecular Systems
Werk Tutzing
Bahnhofstrasse 9-15
82327 Tutzing
Germany
Ludwig-Maximilians-Universitat Munchen
Gene Center/Institute of Biochemistry
Feodor-Lynen-Str.25
81377 Munchen
Germany
The first edition was published under the title
Nonradioactive Labeling and Detection of Biomolecules
(Springer Laboratory)
ISBN 978-3-540-64601-3 ISBN 978-3-642-57206-7 (eBook)
DOI 10.1007/978-3-642-57206-7
Library of Congress Cataloging·in·Publieation Data
Nonradioactive analysis ofbiomoleeules / Christoph Kessler (ed.). - 2nd ed.
p. em. - (Springer lab manual)
Prev. ed published with title: Nonradioactive labeling and detection of biomolecules.
Includes bibliographieal referenees and index.
ISBN 978·3·540-64601-3
1. Biomolecules - Analysis - Laboratory manuals. 1. Kessler, Christoph. II. Series.
QP519.7.N662000
572'.36-dc21
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Preface to the Second Edition
After publication of the first edition of this monograph many of the de
scribed nonradioactive labeling and detection methods have been updated
and successfully adapted to a variety of basic, medical and biotechnology
applications of the life sciences. Especially within the medical field the in
creasing numerous nonradioactive protocols resulted in an accelleration of
basic and clinical research as well as a rapid increase of various applications
using nonradioactive methods which are based on both hybridization tech
niques and on the sequence information obtained by large scale sequencing.
During in the last seven years the Human Genome Project - with the aim of
getting sequence information on the whole human genome - markedly sti
mulated the development of novel nonradioactive technologies and gave rise
to many new methods for nucleic acid analysis. In the period of a few years,
these new methods became standard techniques not only in molecular biol
ogy research laboratories but also clinical and governmental laboratories
working on finding, characterizing, cloning, expression and functional char
acterization of many genes. Furthermore these new methods resulted in a
large number of protocols for diagnosis and mutation analysis of disease
related human genes, and also in novel applications like fingerprint analyses
for parental identification or for clearing up of crime. In addition, the gen
omes of a variety of bacterial species as well as eukaryotic plants and animals
are being analyzed with increasing efforts.
Due to the selectivity and cooperativity of base pairing, nucleic acid ana
lysis facilitates high levels of specificity and sensitivity and even sequence
specific detection of single molecules by means of integration of highly effi
cient amplification reactions into the overall analytic reaction schemes. The
breadth and flexibility of the analytical techniques available is the basis of a
number of medical and biotechnological approaches, such as diagnosis of
infectious diseases (detection of viruses, bacteria or funghi), transplantation
diagnostics (histocompatibility analysis), forensic and veterinary medicine
(fingerprint analysis), plant breeding (resistance pattern analysis), analyses
in the fields of foodstuffs and the environment (pathogenicity tests), produc
tion of recombinant pharmaceutically effective human proteins (specificity
analysis) and monitoring functions in genetic laboratories (security tests).
VIII Preface to the Second Edition
Use of molecular methods in the medical sector not only enhances diag
nostic and therapeutic potentials for infections diseases, but also leads far
beyond this to completely new approaches in Molecular Medicine focussed
in oncology and genetic diseases.
The most important goal of Molecular Medicine is the identification of spe
cific genetic alterations as causes of resulting oncopathological manifesta
tions and hereditary diseases but also responsible for persistent infections.
Knowledge of the molecular basis of these diseases at the sequence level is
the prerequisite for subsequent development of causal genetic diagnosis
and genetic therapy concepts. The Human Genome Project, scheduled for
15 years from the beginning of the nineties and aiming the exhaustive deter
mination of the sequential structure of the human genome, forms the con
ceptual basis of such knowledge.
The second edition of this monograph was updated for many protocol im
provements and a variety of new applications in nonradioactive analysis of
biomolecules especially useful for applications in Molecular Medicine as well
as other applications in the above described fields of basic research and ap
plications in the medical or biotechnology sector. This includes a markedly
broadened application section including e.g. new protocols and methods for
in situ hybridization, sequencing, hybridization on chips, mass spectroscopy
or quantitative real-time amplification formats like the 5' -nuclease (TaqMan)
format. However, also current nonradioactive methods were updated with
new protocols and actual references were included. In addition, a new appen
dix was included with a list of over 1000 application-oriented literature for use
of the nonradioactive digoxigenin system. This reference list is available on
the Springer server (www.springer.de/books/tocl3540646019-ac.pdf).
Thus the second edition of this monograph contains not only the various
methods for nonradioactive labeling and detection of biomolecules but was
broadened to a more complete set of nonradioactive methods for analysis of
biomolecules with the focus on nucleic acids.
I am most indebted to the many authors giving contributions to this mono
graph. Their excellent efforts to write completely new chapters or updating
their contributions from the first edition give rise to a monograph including
actual protocols and information for nonradioactive analysis of bio
molecules. I also wish to express my gratitude to Jutta Lindenborn and
Springer-Verlag for proposing this second edition and giving publishing sup
port; Monika Ortmann and Annemarie Will for superb secretarial skills.
Finally I want to thank all my colleagues and partners from industry and
university for their contributions on new methods and applications of non
radioactive bioanalytics.
Icking-Dorfen, Spring 1999 CHRISTOPH KESSLER
Contents
Introduction
Chapter 1
General Aspects of Nonradioactive Analysis of Biomolecules
CHRISTOPH KESSLER ...........•....••...•••...••..•••.. 1
Part A Standard Nonradioactive labeling Systems
Chapter 2
Overview of Nonradioactive Labeling Systems
CHRISTOPH KESSLER ....••.....•....•....•••....••..•••. 41
The DIG (Digoxigenin:Anti-Digoxigenin) System
Chapter 3
Overview on the Digoxigenin:Anti-Digoxigenin (DIG) System
CHRISTOPH KESSLER .••..•..••...••••..•••••.•••••..•••• 59
Chapter 4
Labeling and Detection of Nucleic Acids
HANS-JOACHIM HOLTKE, KELLY KING, CATHY HOOVER,
IRMGARD OBERMAIER, BARBARA RUGER, KLAUS MUHLEGGER,
AND CHRISTOPH KESSLER 61
Chapter 5
Labeling and Detection of Proteins and Glycoproteins
ANTON HASELBECK AND WOLFGANG HOSEL . . . . . . . • . . . . . • • • .. 94
The Biotin (Biotin:Streptavidin) System
Chapter 6
Labeling and Detection of Nucleic Acids
AYOUB RASHTICHIAN AND JESSE MACKEY ..••••...•••••..... 111
X Contents
Chapter 7
Labeling of Proteins During In Vitro Translation
THOMAS METZLER AND HANS-JOACHIM HOELTKE .......•..... 137
Chapter 8
Labeling and Detection of Proteins and Glycoproteins
EDWARD A. BAYER AND MEIR WILCHEK . . . . . . . . . . . . . . .. •. . .1 5. 5
5-BrdU, AAF, Gold Labeling
Chapter 9
In Vivo Labeling of DNA Probes with 5-BrdU
JEAN-LUC GUESDON . . . . . . . . . . . . . . . . . • . . . . . . . . . . • . . . . . ... 168
Chapter 10
AAF-Iabeling of DNA and Oligonucleotides
JEAN-LUC GUESDON, JACQUES AURIOLA, AND DANIELE CHEVRIER 180
Chapter 11
Colloidal Gold as a Marker in Molecular Biology:
The Use of Ultra-Small Gold Conjugates
PETER F. E. M. VAN DE PLAS AND JAN L. M. LEUNISSEN . . . • . . . .. 193
Direct Enzyme Label
Chapter 12
Direct Peroxidase Labeling of Hybridization Probes
and Chemiluminescence Detection
IAN DURRANT .. . .... . ..... . ........................... 206
Chapter 13
The SNAP System
JAMES E. MARICH AND JERRY L. RUTH 216
Part B Specialized Nonradioactive Detection Systems
Chapter 14
Overview on Nonradioactive Detections Systems
CHRISTOPH KESSLER .................................... 227
Contents XI
Chapter 15
Overview of Colorimetric, Chemiluminometric,
and Fluorimetric Detection Systems
HANS-JOACHIM GUDER, DIETER HEINDL, AND HANS-PETER JOSEL 240
Colorimetric Systems
Chapter 16
Indigo / Tetrazolium Dyes
HANS-JOACHIM GUDER, DIETER HEINDL, AND HANS-PETER JOSEL 249
Chapter 17
Azo Dyes
PETER SCHUSSLER, CHRISTOPH G. GREVELDING,
AND WERNER KUNZ . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .. . .2 5. 3. . .
Luminescence Systems
Chapter 18
Chemiluminescent Detection with Horseradish Peroxidase
and Luminol
DIETER HEINDL, HANS-PETER JOSEL ........................ 258
Chapter 19
Chemiluminescence:
Properties of 1,2-Dioxetane Chemiluminescence
IRENA BRONSTEIN AND LARRY KRICKA ..................... 262
Chapter 20
Electrochemiluminescence: Ruthenium Complexes
JOHN H. KENTEN ....................................... 271
Chapter 21
Bioluminescence: D-Luciferin as Substrate
DUSICA GABRIJELCIC-GEIGER, WERNER MISKA,
AND REINHARD ERICH GEIGER 276
Fluorescence Systems
Chapter 22
Labeling of Biomolecules with Fluorophores
DIETER HEINDL, HANS-PETER JOSEL ..... . . . . . . . . . . . . . . . . . .. 284
