Fluorescent Energy Transfer Nucleic Acid Probes

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.

Includes supplementary material: sn.pub/extras

Klappentext
Although fluorescent probes and assays, which use energy transfer (ET) for monitoring DNA reactions, have multiplied in recent years, until now there have been no manuals summarizing the many different protocols and probe designs. Fluorescent Energy Transfer Nucleic Acid Probes: Designs and Protocols is the first publication to provide such a collection. In the volume, hands-on experts-often the original developers of the technique-comprehensively describe a variety of fluorescent probes and devices that use ET, including molecular beacons, molecular break lights, TaqMan® probes, LUX™ primers, Invader® assay, aptazymes, DNAzymes, molecular machines, biosensors, and logic gates for molecular-scale computation. Merging work on nanotechnology and fluorescent probes, the authors assemble the first comprehensive treatment of all the key DNA- and RNA-based ET probes and present strategies for their optimized custom design. They discuss both fluorescence resonance energy transfer (FRET)-based and non-FRET-based constructs, and provide a complete set of techniques to monitor DNA and RNA reactions, such as hybridization, amplification, cleavage, folding, and associations with proteins, other molecules, and metal ions. Detailed protocols are provided for distance determination in protein-DNA complexes and the detection of topological DNA alterations, mutations, and single-nucleotide polymorphisms. The protocols follow the successful Methods in Molecular Biology™ series format, each offering an outline of the principles behind the technique, step-by-step detailed instructions, and comprehensive troubleshooting.
Exhaustive and state-of-the-art, Fluorescent Energy Transfer Nucleic Acid Probes: Designs and Protocols is an invaluable resource for both novice and experienced researchers who wish to use the latest developments in fluorescent probes in any field of molecular biology.

Inhalt
Design of Energy Transfer Probes.- Selection of Fluorophore and Quencher Pairs for Fluorescent Nucleic Acid Hybridization Probes.- Choosing Reporter-Quencher Pairs for Efficient Quenching Through Formation of Intramolecular Dimers.- Energy Transfer Probes for DNA and RNA Hybridization Detection and Monitoring.- Detection of DNA Hybridization Using Induced Fluorescence Resonance Energy Transfer.- Detecting RNA/DNA Hybridization Using Double-Labeled Donor Probes With Enhanced Fluorescence Resonance Energy Transfer Signals.- Energy Transfer Probes for DNA Breaks Detection and DNA Cleavage Monitoring.- Oscillating Probe for Dual Detection of 5'PO4 and 5'OH DNA Breaks in Tissue Sections.- Using Molecular Beacons for Sensitive Fluorescence Assays of the Enzymatic Cleavage of Nucleic Acids.- A Continuous Assay for DNA Cleavage Using Molecular Break Lights.- Monitoring of DNA Synthesis and Amplification Using Energy Transfer Probes.- Homogenous Detection of Nucleic Acids Using Self-Quenched Polymerase Chain Reaction Primers Labeled With a Single Fluorophore (LUX™ Primers).- Use of Self-Quenched, Fluorogenic LUX™ Primers for Gene Expression Profiling.- TaqMan® Reverse Transcriptase-Polymerase Chain Reaction Coupled With Capillary Electrophoresis for Quantification and Identification of bcr-abl Transcript Type.- Quantitative TaqMan® Assay for the Detection and Monitoring of Cytomegalovirus Infection in Organ Transplant Patients.- Real-Time Detection and Quantification of Telomerase Activity Utilizing Energy Transfer Primers.- DNA Sequence Analysis and Mutation Detection Using Fluorescence Energy Transfer.- Invader® Assay for Single-Nucleotide Polymorphism Genotyping and Gene Copy Number Evaluation.- Real-Time Quantitative Polymerase Chain Reaction Analysis of MitochondrialDNA Point Mutation.- Multiplex Single-Nucleotide Polymorphism Detection by Combinatorial Fluorescence Energy Transfer Tags and Molecular Affinity.- High-Throughput Genotyping With Energy Transfer-Labeled Primers.- Determination of Distance and DNA Folding.- Distance Determination in Protein-DNA Complexes Using Fluorescence Resonance Energy Transfer.- Multi-Fluorophore Fluorescence Resonance Energy Transfer for Probing Nucleic Acids Structure and Folding.- Dna-Based Biosensors Utilizing Energy Transfer.- Fluorescent DNAzyme Biosensors for Metal Ions Based on Catalytic Molecular Beacons.- Fluorescent Energy Transfer Readout of an Aptazyme-Based Biosensor.- Fluorescence Resonance Energy Transfer in the Studies of Guanine Quadruplexes.- Solution-Phase Molecular-Scale Computation With Deoxyribozyme-Based Logic Gates and Fluorescent Readouts.