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Summary
Summary
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 surpr- ingly, these luminous, multicolored probes carry more flashy names than their counterparts in the other fields of molecular biology. In recent years, fluor- 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 p- 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 fluor- cent probes capable of energy transfer. Energy transport within the probe can occur via the resonance energy transfer mechanism, also called Förster tra- 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.
Table of Contents
| Preface | p. v |
| Contributors | p. xiii |
| Companion CD | p. xv |
| Part I Design of energy transfer probes | |
| 1 Selection of Fluorophore and Quencher Pairs for Fluorescent Nucleic Acid Hybridization Probes | p. 3 |
| 2 Choosing Reporter-Quencher Pairs for Efficient Quenching Through Formation of Intramolecular Dimers | p. 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 | p. 33 |
| 4 Detecting RNA/DNA Hybridization Using Double-Labeled Donor Probes With Enhanced Fluorescence Resonance Energy Transfer Signals | p. 43 |
| Part III Energy Transfer Probes for DNA Breaks Detection and DNA cleavage monitoring | |
| 5 Oscillating Probe for Dual Detection of 5'PO 4 and 5'OH DNA Breaks in Tissue Sections | p. 59 |
| 6 Using Molecular Beacons for Sensitive Fluorescence Assays of the Enzymatic Cleavage of Nucleic Acids | p. 71 |
| 7 A Continuous Assay for DNA Cleavage Using Molecular Break Lights | p. 83 |
| 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 (LUX TM Primers) | p. 95 |
| 9 Use of Self-Quenched, Fluorogenic LUX TM Primers for Gene Expression Profiling | p. 115 |
| 10 TaqMan" Reverse Transcriptase-Polymerase Chain Reaction Coupled With Capillary Electrophoresis for Quantification and Identification of bcr-abl Transcript Type | p. 135 |
| 11 Quantitative TaqMan" Assay for the Detection and Monitoring of Cytomegalovirus Infection in Organ Transplant Patients | p. 147 |
| 12 Real-Time Detection and Quantification of Telomerase Activity Utilizing Energy Transfer Primers | p. 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 | p. 173 |
| 14 Real-Time Quantitative Polymerase Chain Reaction Analysis of Mitochondrial DNA Point Mutation | p. 187 |
| 15 Multiplex Single-Nucleotide Polymorphism Detection by Combinatorial Fluorescence Energy Transfer Tags and Molecular Affinity | p. 201 |
| 16 High-Throughput Genotyping With Energy Transfer-Labeled Primers | p. 215 |
| Part VI Determination of Distance and DNA Folding | |
| 17 Distance Determination in Protein-DNA Complexes Using Fluorescence Resonance Energy Transfer | p. 243 |
| 18 Multi-Fluorophore Fluorescence Resonance Energy Transfer for Probing Nucleic Acids Structure and Folding | p. 257 |
| Part VII DNA-Based Biosensors Utilizing Energy Transfer | |
| 19 Fluorescent DNAzyme Biosensors for Metal Ions Based on Catalytic Molecular Beacons | p. 275 |
| 20 Fluorescent Energy Transfer Readout of an Aptazyme-Based Biosensor | p. 289 |
| 21 Fluorescence Resonance Energy Transfer in the Studies of Gua9 Quadruplexes | p. 311 |
| 22 Solution-Phase Molecular-Scale Computation With Deoxyribozyme-Based Logic Gates and Fluorescent Readouts | p. 343 |
| Index | p. 365 |
