Fluorescence quenching detection assay

Kinetic Fluorescence-Quenching Detection Assay for Allele Frequency Estimation... 

Although a number of quantitative assays are available for allele frequency estimation, most are difficult and expensive to develop or implement. In this chapter, we describe the most versatile and least expensive assays for allele frequency estimation, namely, the template-directed dye-terminator incorporation assay with fluorescence quenching detection (the FQ-TDI assay [3]). The FQ-TDI assay is a real-time homogeneous primer extension assay based on two principles, namely, that deoxyribonucleic acid (DNA) polymerase catalyzes the allele-specific incorporation of a dye-terminator at the polymorphic site and that the fluorescence intensities of a fluorescent dye decreases significantly when it is incorporated into primers. 

Brown et al.68 have developed a cellulose plate with a fluorescent indicator. Compounds are developed in 3.0% (w/v) NHfc.Cl and detected by fluorescence quenching. These authors also use 0.5% mercaptoethanol in their mobile phase, but this is only to prevent oxidation of the labile reduced pteridines, which are not adequately protected by substitution at the N5 position. Since neutral or alkaline solutions of leucovorin are relatively stable in air, this precaution may not be required for routine assay. 

Kinase substrates can become resistant to the actions of proteases due to their phosphorylations. Thus, the fluorescence quench assays (described in Chapter 2 covering protease assays) can be used to measure kinase activity. The assays can be viewed as coupled because they require a second enzyme to convert a product or substrate into a detectable signal. With kinase assays, the formation of phosphopeptide inhibits the protease action on the peptide and the signal remains quenched and therefore decreased (Rodems et al., 2002). Inhibiting the kinase results in increases in protease sensitivity and in signal. 

Donor-acceptor probes are very useful as fluorescent lables in biochemical assay and sensing based on the fluorescence quenching mechanisms. In a closed state form where the donor and acceptor are close to each other, the fluorescence of the donor (reporter) is highly quenched. In an open state, where the donor and receptor are spaced away from each other due to biochemical reactions, the donor fluorescnece is restored. The changes of the fluorescence intensity of the reporter has been used for DNA detection, immunoassay, enzyme sensing and detection of many other bimolecules. 

Fluorescence intensity assays detect an increase or a decrease in the strength of a fluorescence emission. Based on the origin of this change in fluorescence, the two main classes are fluorogenic assays and fluorescence quench assays. 

Among the first modern indicator displacement assays in supramolecular chemistry was the fluorescence-based detection of the neurotransmitter acetylcholine developed by Inouye, Scheme 11.7.31 The fluorescence of the pyrene derivative is quenched by binding to a deprotonated... 

A EXPERIMENTAL FIGURE 18-5 In vitro fluorescence quenching assay can detect phospholipid flippase activity of... 

Recently another approach for the detection of protease activity by using fluorescent quenching of a conjugated polyelectrolyte was reported [52] (Figure 13.9). This assay utilizes a covalent attachment of the quencher-labeled peptide substrate to the anionic-conjugated polyelectrolyte and combines the... 

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