Protocols for Nucleic Acid Analysis by Nonradioactive Probes 2nd Ed (Methods in Molecular Biology Vol 353)

334 Pages·2007·3.99 MB·English

by  Elena

METHODS IN MOLECULAR BIOLOGY™ 353
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Protocols for Nucleic Acid Analysis
by Nonradioactive Probes, Second Edition
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M E T H O D S I N M O L E C U L A R B I O L O G Y™
Protocols for Nucleic
Acid Analysis
by Nonradioactive
Probes
Second Edition
Edited by
Elena Hilario
Gene Technologies, HortResearch Ltd.
Mt. Albert, Auckland, New Zealand
John Mackay
Applied Science, Roche Diagnostics
Auckland, New Zealand
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eISBN 1-59745-229-7 • 978-1-59745-229-8
ISSN 1064-3745
Library of Congress Cataloging-in-Publication Data
Protocols for nucleic acid analysis by nonradioactive probes. — 2nd ed. / edited by Elena Hilario, John
Mackay.
p. ; cm. — (Methods in molecular biology, ISSN 1064-3745 ; 353)
Includes bibliographical references and index.
ISBN 1-58829-430-7 (alk. paper)
1. Nucleic acid probes. 2. Nucleic acids—Analysis. I. Hilario, Elena, 1966- II. Mackay, John, 1969-
III. Series: Methods in molecular biology (Clifton, N.J.) ; 353.
[DNLM: 1. DNA—analysis. 2. DNA Probes. 3. Molecular Probe Techniques. 4. Nucleic Acid
Amplification Techniques. W1 ME9616J v.353 2006 / QU 58.5 P9674 2006]
QP620.P765 2006
572.8’636—dc22
2006003449
Preface
Since Protocols for Nucleic Acid Analysis by Nonradioactive Probes was
published in 1994, the use of techniques such as Southern and Northern blotting has
continued unabated, despite the continuing explosion of polymerase chain reaction
(PCR)-based techniques that have replaced many traditional molecular biology meth-
ods. Indeed, PCR is now frequently used with nonradioactive probe formats in appli-
cations such as real-time PCR that may be likened to an “online” Southern or Northern
blot!
More often than is realized, radioactive techniques are not essential for the analysis
of nucleic acids in molecular biology. Nonradioactive methods have been available
for the scientific community for more than 20 years, providing excellent results. Sci-
entists who rely on nonradioactive techniques have indirectly promoted the develop-
ment of new nonradioactive haptens, substrates, means of detection, and a wide range
of novel applications. These improvements on the detection limits of nonradioactive
methods can easily compete with radioactive protocols, which are now seen as slow,
cumbersome, and only required in very specific experimental designs.
Protocols for Nucleic Acid Analysis by Nonradioactive Probes, Second Editionaims
to provide a firm background on the basic preparative protocols required for the analy-
sis of nucleic acids by nonradioactive methods, as well as presenting the methodolo-
gies using these amazing new applications. This volume offers guide chapters on
nucleic acid extractions, preparation of nucleic acid blots, and labeling of nucleic
acids with nonradioactive haptens. There are two ways of detecting nonradioactive
probes: by indirect methods using a labeled probe or by directly detecting the labeled
nucleic acid. We have divided the contents accordingly. These are key examples of the
extensive potential that nonradioactive detection provides to the molecular biology
community. Our target audience is not limited to the research laboratory only; we
hope that tertiary students and post-doctorates will find the content of this book a
useful reference guide in their projects.
Elena Hilario
John Mackay
v

Contents
Preface ..............................................................................................................v
Contributors .....................................................................................................ix
PART I. NUCLEIC ACID EXTRACTIONS
1 Genomic DNA Isolation From Different Biological Materials
Duckchul Park.......................................................................................3
2 Extraction of Plant RNA
Elspeth MacRae...................................................................................15
PART II. INDIRECT DETECTION
3 Overview of Hybridization and Detection Techniques
Elena Hilario.......................................................................................27
4 Checkerboard DNA–DNA Hybridization Technology
Using Digoxigenin Detection
Lisa S. Gellen, Glenn M. Wall-Manning, and Chris H. Sissons..........39
5 Nonradioactive Northern and Southern Analyses
From Plant Samples
Christoph Peterhaensel, Dagmar Weier, and Thomas Lahaye...........69
6 Screening a BAC Library With Nonradioactive Overlapping
Oligonucleotide (Overgo) Probes
Elena Hilario, Tiffany F. Bennell, and Erik Rikkerink.........................79
7 Direct In-Gel Hybridization of DNA With
Digoxigenin-Labeled Probes
Saeed A. Khan and Mohamed S. Nawaz.............................................93
8 In Situ Hybridization of Termite Microbes
Shigeharu Moriya, Satoko Noda, Moriya Ohkuma,
and Toshiaki Kudo........................................................................105
PART III. FLUORESCENT LABELING AND DETECTION
9 RNA Electrophoretic Mobility Shift Assay Using a Fluorescent
DNA Sequencer
Yukinori Eguchi.................................................................................115
vii
viii Contents
10 Comparative Quantitation of mRNA Expression
in the Central Nervous System Using Fluorescence
In SituHybridization
Darren J. Day, Eli M. Mrkusich, and John H. Miller........................125
11 Visualization of Gene Expression by Fluorescent Multiplex
Reverse Transcriptase-PCR Amplification
María Rosa Ponce, Víctor Quesada, Andrea Hricová,
and José Luis Micol.......................................................................143
12 Fluorescence In Situ Hybridization for the Identification
of Environmental Microbes
Annelie Pernthaler and Jakob Pernthaler.........................................153
PART IV. KINETIC (“REAL-TIME”) PCR
13 Introduction to Kinetic (Real-Time) PCR
John Mackay.....................................................................................167
14 Validation of Short Interfering RNA Knockdowns by Quantitative
Real-Time PCR
Sukru Tuzmen, Jeff Kiefer, and Spyro Mousses................................177
15 Real-Time Quantitative PCR as an Alternative to Southern Blot
or Fluorescence In Situ Hybridization for Detection
of Gene Copy Number Changes
Jasmien Hoebeeck, Frank Speleman, and Jo Vandesompele............205
16 Design and Work-Up of a New Molecular Diagnostic Assay
Based on Real-Time PCR
Harald H. Kessler..............................................................................227
17 Real-Time PCR Fluorescent Chemistries
John Mackay and Olfert Landt.........................................................237
PART V. MICROARRAYS
18 Microarrays: An Overview
Norman H. Lee and Alexander I. Saeed...........................................265
19 Oligonucleotide Microarrays for the Study of Coastal
Microbial Communities
Gaspar Taroncher-Oldenburg and Bess B. Ward.............................301
Index ............................................................................................................317
Contributors
TIFFANY F. BENNELL • Gene Technologies, HortResearch Ltd., Mt. Albert, Auckland,
New Zealand
DARREN J. DAY • School of Biological Sciences, Victoria University, Wellington,
New Zealand
YUKINORI EGUCHI • Research Laboratory, Faculty of Medicine, University
of Ryukyus, Okinawa, Japan
LISA S. GELLEN • Dental Research Group, Department of Pathology and Molecular
Medicine, Wellington School of Medicine and Health Sciences, University
of Otago, Wellington, New Zealand
ELENA HILARIO • Gene Technologies, HortResearch Ltd., Mt. Albert, Auckland,
New Zealand
JASMIEN HOEBEECK • Center for Medical Genetics, Ghent University Hospital,
Ghent, Belgium
ANDREA HRICOVÁ • División de Genética and Instituto de Bioingeniería, Universidad
Miguel Hernández, Campus de Elche, Alicante, Spain
HARALD H. KESSLER • Institute of Hygiene, Medical University of Graz, Austria
SAEED A. KHAN • Division of Microbiology, National Center for Toxicological
Research, U.S. Food and Drug Administration, Jefferson, AR
JEFF KIEFER • Knowledge Mining Laboratory, Pharmaceutical Genomics Division,
Translational Genomics Research Institute, Scottsdale, AZ
TOSHIAKI KUDO • Environmental Molecular Biology Laboratory, RIKEN Institute;
Graduate School of Yokohama City University, Yokohama City, Japan
THOMAS LAHAYE•Martin-Luther-University Halle-Wittenberg, Institute for Genetics,
Halle, Germany
OLFERT LANDT • TIB MOLBIOL Syntheselabor, Berlin, Germany
NORMAN H. LEE • Department of Functional Genomics, The Institute for Genomic
Research, Rockville, MD
JOHN MACKAY • Applied Science, Roche Diagnostics, Mt. Wellington, Auckland,
New Zealand; Current Address: Linnaeus Laboratory, Gisborne, New Zealand
ELSPETH MACRAE • Biomaterials Research, Scion, Rotorua, New Zealand; Gene
Technologies, HortResearch Ltd., Mt. Albert, Auckland, New Zealand
JOSÉ LUIS MICOL • División de Genética and Instituto de Bioingeniería, Universidad
Miguel Hernández, Campus de Elche, Alicante, Spain
JOHN H. MILLER • School of Biological Sciences, Victoria University, Wellington,
New Zealand
SHIGEHARU MORIYA•Environmental Molecular Biology Laboratory, RIKEN Institute;
Graduate School of Yokohama City University, Yokohama City, Japan
ELI M. MRKUSICH • School of Biological Sciences, Victoria University, Wellington,
New Zealand
ix