Fluorescence resonance energy transfer FRET assays

Ledford M, Friedman KD, Hessner MJ, Moehlenkamp C, Williams TM, Larson RS. A multi-site study for detection of the factor V (Leiden) mutation from genomic DNA using a homogeneous invader microtiter plate fluorescence resonance energy transfer (FRET) assay. J Molec Diag 2000 2 97-104. 

Time- and frequency-domain fluorescence methods have been used in different fields for several purposes, such as distinguishing sample constituents whose fluorescence spectra overlap one another, to distinguish the fluorescence of an analyte from background scattering or luminescence of other sample constituents, in combination with fluorescence resonance energy transfer (FRET) assays to combine the benefits of FRET and time-resolved fluorescence. A listing of some selected applications is given in Table 3. 

Compounds that interfere with the detection mechanism of the HTS assay wiU, in many cases, be detected as highly potent actives [31]. One example would be compounds that intrinsically emit or absorb light at the wavelengths used in a fluorescence-based assay such as fluorescence resonance energy transfer (FRET). In an HTS screen using a fluorescence-based assay at Wyeth, 1.2% of the samples tested showed not just high fluorescence but the maximum possible initial (time 0) reading on the fluorimeter. 

As the investigation of the interactions between H DAC inhibitors and the enzymes are an important issue, competition assay systems are helpful implements in facilitating the characterization of inhibitor binding. Such a competition binding assay that has been developed for histone deacetylases is based on fluorescence resonance energy transfer (FRET) between tryptophan residues of the histone deacetylase and a fluorescent HDAC inhibitor [38]. In competition with other... 

While the capture on DNA chips of fluorophore-labelled targets, and the extension of arrayed primers with fluorophore-labelled nucleotides has been widely used for some time, it is only more recently that assay formats have developed that utilize immobilized nucleic acids already modified with fluorophores. Fundamental analyses of surface monolayer structures and chemistries can be readily performed by immobilizing such modified oligonucleotides into SAM structures [105,106], but it is those interactions that can be monitored using fluorescence quenching or fluorescence resonance energy transfer (FRET) that have gained the most attention. 

Since the initial paper by Fields and Song, there have been significant technical improvements in the method. DNA-binding domains and transcription activation domains have been optimized to reduce false positives and increase the transcription read-out. A variety of reporter plasmids have been engineered to detect a broad range of protein-protein interactions. Much more is understood about the nature of false positives and how to rout them out. Moreover, in response to the utility of this approach, several laboratories have begun to develop transcription-based assays that can be carried out in bacteria, or protein-protein interaction assays based on alternate readouts such as enzyme complementation or fluorescence resonance energy transfer (FRET). 

Figure 37-24 Common probes and dyes for real-time PCR. f/J Double-stranded DNA dyes show a significant increase in fluorescence when bound to DNA (hv = excitation light). (2) Adjacent hybridization probes. Fluorescence resonance energy transfer (FRET) is illustrated between a donor and acceptor fluorophore.The x indicates phosphorylation of the 3 terminus of the probe to prevent polymerase extension. (3) FRET between a labeled primer and a single hybridization probe. (4) Hydrolysis probes are cleaved between the reporter and quencher, resulting in increased fluorescence. (5) Hairpin probes are quenched in the native conformation, but increase in fluorescence when hybridized. (6) Hairpin primers retain their native, quenched conformation until they are incorporated into a double-stranded product (Modified with permission of the publisher from Pritham GH, Wittwer CT Continuous f/uorescent monitoring of PCR.J Clin Ug Assay 1998, 21 404-412. 1998 Clinical Ligand Assay Society, Inc.)...

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