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 elevated in recent times, in the past there were no manuals summarizing the numerous various protocols and probe designs. Fluorescent power move Nucleic Acid Probes: Designs and Protocols is the 1st e-book to supply any such assortment. within the quantity, hands-on experts-often the unique builders of the technique-comprehensively describe numerous 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 good judgment gates for molecular-scale computation. Merging paintings on nanotechnology and fluorescent probes, the authors gather the 1st accomplished therapy of all of the key DNA- and RNA-based ET probes and current suggestions for his or her optimized customized layout. They speak about either fluorescence resonance power move (FRET)-based and non-FRET-based constructs, and supply a whole set of options 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 decision in protein-DNA complexes and the detection of topological DNA changes, mutations, and single-nucleotide polymorphisms. The protocols persist with the profitable tools in Molecular Biology™ sequence layout, every one delivering an overview of the foundations at the back of the process, step by step distinct directions, and complete 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 newest advancements in fluorescent probes in any box of molecular biology.
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Extra resources for Fluorescent Energy Transfer Nucleic Acid Probes: Designs and Protocols
2). Static quenching involves formation of an intramolecular dimer; the dyes aggregate and stick together. Dye aggregation in aqueous solvents is controlled by electrostatic, steric, and hydrophobic forces (2). 4. Cyanine (Cy3, Quasar 670, and others) and rhodamine (Cal Orange, Cal Red, ROX, and so on) dyes are quite planar, hydrophobic, and have delocalized charge because of quaternary nitrogens. , FAM, JOE, TET, Cal Gold, HEX) (Figs. 3–5). 2. Measuring Quenching Efficiencies See Notes 3–5 and Fig.
Introduction Fluorescence resonance energy transfer (FRET) occurs when two fluorophores are in close proximity and the emission spectrum of one probe overlaps the excitation spectrum of the other. The emission energy of a donor fluorophore is transferred to excite an acceptor fluorophore (1,2). A set of DNA probes, hybridized next to one another on target RNA (messenger RNA [mRNA], viral RNA), can be labeled with different fluorophores for the detection of specific RNA both in vitro (3–5) and in vivo (6,7).
Presents a protocol for immobilizing the target to a streptavidin-coated microtiter plate. 3. 4. explains how to collect and examine the fluorescence data. 1. Target Amplification Via PCR 1. To simplify PCR set-up, it is recommended to dry the genomic DNA (10 ng) to the bottom of one of the wells of the microtiter plate (see Note 2). 2 mM of each dNTP, with the remainder volume consisting of double-distilled H2O. 2. Temperature cycling should consist of an initial 10 min incubation at 94°C to activate the Taq polymerase, followed by 40 cycles of denaturation at 94°C for 15 s and annealing/elongation at the appropriate annealing temperature for the primer pair (see Note 5) for 30 s.