Fluorescent Energy Transfer Nucleic Acid Probes: Designs and Protocols (Methods in Molecular Biology, 335)

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.

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.