By Salvatore A. E. Marras (auth.), Vladimir V. Didenko MD, PhD (eds.)
Although fluorescent probes and assays, which use strength move (ET) for tracking DNA reactions, have accelerated lately, before there were no manuals summarizing the numerous diverse protocols and probe designs. Fluorescent strength move Nucleic Acid Probes: Designs and Protocols is the 1st ebook to supply this type of assortment. within the quantity, hands-on experts-often the unique builders of the technique-comprehensively describe a number of fluorescent probes and units that use ET, together with molecular beacons, molecular holiday lighting fixtures, TaqMan® probes, LUX™ primers, Invader® assay, aptazymes, DNAzymes, molecular machines, biosensors, and common sense gates for molecular-scale computation. Merging paintings on nanotechnology and fluorescent probes, the authors gather the 1st finished remedy of the entire key DNA- and RNA-based ET probes and current innovations for his or her optimized customized layout. They talk about either fluorescence resonance power move (FRET)-based and non-FRET-based constructs, and supply a whole set of innovations to watch DNA and RNA reactions, similar to hybridization, amplification, cleavage, folding, and institutions with proteins, different molecules, and steel ions. specific protocols are supplied for distance choice in protein-DNA complexes and the detection of topological DNA changes, mutations, and single-nucleotide polymorphisms. The protocols keep on with the profitable tools in Molecular Biology™ sequence layout, every one providing an summary of the foundations in the back of the approach, step by step unique directions, and finished troubleshooting.
Exhaustive and state of the art, Fluorescent strength move Nucleic Acid Probes: Designs and Protocols is a useful source for either beginner and skilled researchers who desire to use the most recent advancements in fluorescent probes in any box of molecular biology.
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Additional info for Fluorescent Energy Transfer Nucleic Acid Probes: Designs and Protocols
3. Discard the solution, and add 50 µL of HEN buffer containing a 1:10,000 dilution of the SYBR Green I (the iFRET donor) directly from the tube as supplied by the manufacturer (see Note 10). 4. Detection of Melting Curves Using iFRET 1. 3°C per second. 2. Export the data file to a spreadsheet (such as Microsoft Excel®) and extract the appropriate fluorescence and temperature data (see Notes 11 and 12). 3. A plot of the fluorescence vs temperature data produces a characteristic melting curve. When the Tm is reached, the probe and target separate and the FRET reaction stops as marked by a large drop in fluorescence (see Note 13 and Fig.
There are several ways to screen combinations of reporter/quencher pairs. A series of quencher–reporter pairings were recently tested by Marras, Kramer, and Tyagi. They used complementary oligos with 5'-reporters and 3'-quenchers to bring the dyes directly together or at staggered distances to measure, respectively, contact-mediated and FRET quenching efficiencies (5) (Fig. 3). This method of bringing the dyes together in order to measure contact (static) quenching holds the reporter and quencher in a fairly constrained and fixed relative orientation.
The labeled oligonucleotides had to be purified with reverse-phase high-performance liquid chromatography. The detection of hybridization is limited by the concentration of the target RNA molecules and sensitivity of the detector devices; thus, relatively abundant samples can be analyzed using a conventional spectrofluorometer (F-5000, Hitachi), whereas more dilute samples should be measured using a fluorescence microscope equipped with a detector device (such as a C-CCD camera: C4880-40, Hamamatsu Photonics) or a confocal laser-scanning microscope system (µ-radiance, Bio-Rad, Hercules, CA).