Table Of ContentFluorescent Energy Transfer Nucleic Acid Probes
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357. Cardiovascular Proteomics: Methods and Proto- 332299. Embryonic Stem Cell Protocols, Second Edition,
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333355.Fluorescent Energy Transfer Nucleic Acid 330088. Tedhiteerda pbyeu Cti.c M Parrokt eSimnsa:l eMs eatnhdo dDs aavnidd CPr. oJtaomcoelss,, 2005
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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™
Fluorescent
Energy Transfer
Nucleic Acid Probes
Designs and Protocols
Edited by
Vladimir V. Didenko, ,
MD PhD
Baylor College of Medicine, Houston, TX
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Cover illustration: Apoptotic rat thymus triple-stained by oscillating probe and fluorescent blue dye DAPI.
Two types of DNA breaks are labeled with green and red fluorophores. Description of the probe is in Chapter
5, "Oscillating Probe for Dual Detection of 5'PO and 5'OH DNA Breaks in Tissue Sections," by Vladimir
4
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Fluorescent energy transfer nucleic acid probes : designs and protocols / edited by Vladimir V. Didenko.
p. ; cm. -- (Methods in molecular biology ; v. 335)
Includes bibliographical references and index.
ISBN 1-58829-380-7 (alk. paper)
1. Nucleic acid probes.
[DNLM: 1. Nucleic Acid Probes. 2. Fluorescence Resonance Energy Transfer--methods. 3. Genetic
Techniques. QU 58 F647 2006] I. Didenko, Vladimir V. II. Series: Methods in molecular biology (Clifton,
N.J.) ; v. 335.
QP624.5.D73F558 2006
572.8'4--dc22
2005023841
Preface
Fluorescent nucleic acid probes, which use energy transfer, include such
constructs as molecular beacons, molecular break lights, Scorpion primers,
TaqMan probes, and others. These probes signal detection of their targets by
changing either the intensity or the color of their fluorescence. Not surpris-
ingly, these luminous, multicolored probes carry more flashy names than their
counterparts in the other fields of molecular biology. In recent years, fluores-
cent probes and assays, which make use of energy transfer, have multiplied at
a high rate and have found numerous applications. However, in spite of this
explosive growth in the field, there are no manuals summarizing different pro-
tocols and fluorescent probe designs. In view of this, the main objective of
Fluorescent Energy Transfer Nucleic Acid Probes: Designs and Protocols is
to provide such a collection.
Oligonucleotides with one or several chromophore tags can form fluores-
cent probes capable of energy transfer. Energy transport within the probe can
occur via the resonance energy transfer mechanism, also called Förster trans-
fer, or by non-Förster transfer mechanisms. Although the probes using Förster
transfer were developed and used first, the later non-Förster-based probes, such
as molecular beacons, now represent an attractive and widely used option. The
term “fluorescent energy transfer probes” in the title of this book covers both
Förster-based fluorescence resonance energy transfer (FRET) probes and
probes using non-FRET mechanisms.
Energy transfer probes serve as molecule-size sensors, changing their
fluorescence upon detection of various DNA reactions. Many types of energy
transfer probes can be adapted for homogenous detection formats, i.e., they
function autonomously and fluorescently indicate their molecular targets with-
out additional intervention. In this case, the energy transfer phenomenon serves
as a “molecular cloaking device,” hiding the probe’s fluorescence until it
detects its target. In the nonreactive state, probe fluorescence is quenched as a
result of energy transfer to a nonfluorescent acceptor located in close proxim-
ity to a fluorophore. After the reaction, relative positions of a fluorophore and
a quencher change, resulting in the appearance of fluorescence.
The DNA-based energy transfer constructs can also form “composition
fluorophores” in which the emission and absorption properties can be indepen-
dently tuned, making them attractive for the multiplex detection assays.
v
vi Preface
Energy transfer probes are especially advantageous when used in multiplex
polymerase chain reaction, as parts of biosensor assays, for screening and real-
time monitoring of biochemical reactions, and in nanotechnology applications.
Fluorescent Energy Transfer Nucleic Acid Probes: Designs and Protocols
presents a wide assortment of methods using both Förster and non-Förster
mechanisms of energy transfer in nucleic acids. A broad array of structures and
applications of various energy transfer constructs and their optimized design
are presented in detail for the first time.
The techniques described include those designed to monitor various types
of DNA and RNA reactions including hybridization, amplification, cleavage,
folding, and association with proteins, other molecules, and metal ions. This
volume also presents techniques for distance determination in protein–DNA
complexes and methods to detect topological DNA alterations, mutations, and
single nucleotide polymorphisms. It contains the latest cutting-edge
nanotechnology applications, such as nanomachines, energy transfer
aptazymes, DNAzyme-based biosensors, and logic gates for molecular-scale
computation.
Reproduction of technical protocols, readily available from original journal
papers, would not warrant an additional publication. Fluorescent Energy Trans-
fer Nucleic Acid Probes: Designs and Protocols instead serves as a compre-
hensive source of information on every method described.
The volume is divided into seven sections consisting of two to five chapters.
The first section contains two chapters describing basic principles of selection
and optimization of labels for FRET-based (Chapter 1) and non-FRET-based
(Chapter 2) probes applicable to all energy transfer constructs. The section
provides information necessary for the individualized design of energy trans-
fer probes considering the specific needs of a researcher. Parts II–VI discuss
application of energy transfer probes for detection and monitoring of various
reactions involving DNA or RNA including: hybridization detection (Chapters
3 and 4), DNA breaks and cleavage monitoring (Chapters 5–7), synthesis and
amplification visualization (Chapters 8–12), sequence analysis and mutation
detection (Chapters 13–16), and determination of distances and DNA folding
(Chapters 17–18). The last section (Chapters 19–22) deals with design and
application of molecular devices that use energy transfer, such as biosensors,
nanomachines, and logic gates for molecular-scale computation. Chapter 5
describes a molecular machine for DNA breaks detection and therefore
belongs to both the nanotechnology and DNA damage fields.
The field of fluorescent probes is constantly evolving and I hope that this volume
will not only help its readers use the described techniques, but will prompt
them to explore new ways in which energy transfer constructs can facilitate
their research.
vii Preface
Researchers using fluorescence in any field of biomedical sciences will ben-
efit from this book. These include molecular and cell biology, embryology,
toxicology, radiobiology, experimental and clinical pathology, oncology,
experimental pharmacology, drug design, environmental science, and
nanotechnology. Fluorescent Energy Transfer Nucleic Acid Probes: Designs
and Protocols is a helpful resource for both novice investigators and experi-
enced researchers. For a scientist new to the area of fluorescent probes, the
book will help to select the suitable probe, to deal with experimental pitfalls
and to properly interpret the results. Experienced researchers will find the book
useful because it describes the new and unique constructs in detail and can be
used as a source of information in development of new energy transfer probes
and applications.
I am grateful to all participating authors whose ingenuity made this book
possible, and particularly to those of them who submitted their contributions
on time. I wish to express my appreciation to Candace Minchew for her expert
technical assistance. I also thank Professor John Walker for his generous help
with the review process.
Vladimir V. Didenko, MD,PhD
Contents
Preface ..............................................................................................................v
Contributors ...................................................................................................xiii
Companion CD...............................................................................................xv
PART I. DESIGN OF ENERGY TRANSFER PROBES
1 Selection of Fluorophore and Quencher Pairs for Fluorescent
Nucleic Acid Hybridization Probes
Salvatore A. E. Marras..........................................................................3
2 Choosing Reporter–Quencher Pairs for Efficient Quenching
Through Formation of Intramolecular Dimers
Mary Katherine Johansson..................................................................17
PART II.ENERGY TRANSFER PROBES FOR DNA AND RNA
HYBRIDIZATION DETECTION AND MONITORING
3 Detection of DNA Hybridization Using Induced Fluorescence
Resonance Energy Transfer
W. Mathias Howell.............................................................................33
4 Detecting RNA/DNA Hybridization Using Double-Labeled
Donor Probes With Enhanced Fluorescence Resonance
Energy Transfer Signals
Yukio Okamura and Yuichiro Watanabe............................................43
PART III. ENERGY TRANSFER PROBES FOR DNA BREAKS DETECTION
AND DNA CLEAVAGE MONITORING
5 Oscillating Probe for Dual Detection of 5’PO and 5’OH DNA
4
Breaks in Tissue Sections
Vladimir V. Didenko...........................................................................59
6 Using Molecular Beacons for Sensitive Fluorescence Assays
of the Enzymatic Cleavage of Nucleic Acids
Chaoyong James Yang, Jeff Jianwei Li, and Weihong Tan.................71
7 A Continuous Assay for DNA Cleavage Using Molecular
Break Lights
John B. Biggins, James R. Prudent, David J. Marshall,
and Jon S. Thorson.........................................................................83
ix
x Contents
PART IV. MONITORING OF DNA SYNTHESIS AND AMPLIFICATION
USING ENERGY TRANSFER PROBES
8 Homogenous Detection of Nucleic Acids
Using Self-Quenched Polymerase Chain Reaction
Primers Labeled With a Single Fluorophore (LUXTM Primers)
Irina Nazarenko..................................................................................95
9 Use of Self-Quenched, Fluorogenic LUXTM Primers
for Gene Expression Profiling
Wolfgang Kusser...............................................................................115
10 TaqMan® Reverse Transcriptase-Polymerase Chain
Reaction Coupled With Capillary Electrophoresis
for Quantification and Identification
ofbcr-abl Transcript Type
Rajyalakshmi Luthra and L. Jeffrey Medeiros...................................135
11 Quantitative TaqMan® Assay for the Detection and Monitoring
of Cytomegalovirus Infection in Organ Transplant Patients
Heli Piiparinen and Irmeli Lautenschlager.......................................147
12 Real-Time Detection and Quantification of Telomerase Activity
Utilizing Energy Transfer Primers
Hiroshi Uehara.................................................................................157
PART V.DNA SEQUENCE ANALYSIS AND MUTATION DETECTION
USING FLUORESCENCE ENERGY TRANSFER
13 Invader® Assay for Single-Nucleotide Polymorphism
Genotyping and Gene Copy Number Evaluation
Andrea Mast and Monika de Arruda................................................173
14 Real-Time Quantitative Polymerase Chain Reaction Analysis
of Mitochondrial DNA Point Mutation
Lee-Jun C. Wong and Ren-Kui Bai....................................................187
15 Multiplex Single-Nucleotide Polymorphism Detection
by Combinatorial Fluorescence Energy Transfer Tags
and Molecular Affinity
Anthony K. Tong and Jingyue Ju.......................................................201
16 High-Throughput Genotyping With Energy
Transfer-Labeled Primers
Yuri Khripin.......................................................................................215
xi Contents
PART VI.DETERMINATION OF DISTANCE AND DNA FOLDING
17 Distance Determination in Protein–DNA Complexes Using
Fluorescence Resonance Energy Transfer
Mike Lorenz and Stephan Diekmann................................................243
18 Multi-Fluorophore Fluorescence Resonance Energy Transfer
for Probing Nucleic Acids Structure and Folding
Juewen Liu and Yi Lu........................................................................257
PART VII. DNA-BASED BIOSENSORS UTILIZING ENERGY TRANSFER
19 Fluorescent DNAzyme Biosensors for Metal Ions Based
on Catalytic Molecular Beacons
Juewen Liu and Yi Lu........................................................................275
20 Fluorescent Energy Transfer Readout
of an Aptazyme-Based Biosensor
David Rueda and Nils G. Walter......................................................289
21 Fluorescence Resonance Energy Transfer in the Studies
of Guanine Quadruplexes
Bernard Juskowiak and Shigeori Takenaka.......................................311
22 Solution-Phase Molecular-Scale Computation
With Deoxyribozyme-Based Logic Gates
and Fluorescent Readouts
Joanne Macdonald, Darko Stefanovic,
and Milan N. Stojanovic...............................................................343
Index............................................................................................................365
