FRET probes types

Fig. 6.11. Two types of FRET probes. (A) Ratiometric probes are formed by two fluorescent molecules that allow determination of emission ratio. (B) Quenched probes feature a donor fluorophore and a quencher. The emission increase of the donor after release of the acceptor is detected. Both types are frequently used to build proteinases probes.

FRET probes have not only been generated to measure the phospholipase activity but to study its substrate specificity as well. Several substrates of PLA2 with a variety of head groups and labeled with a BODIPY dye and a Dabcyl quencher were created by Rose et al. and tested against different PLAs in cells to determine substrate specificity and intracellular localization [137], The specificity of PLA2 isoforms towards the number of double bonds in the sn2 position was evaluated with a small series of PENN derivatives. It was demonstrated that the cytosolic type V PLA2 preferred substrates with a single double bond [138],... 

Fig. 14. Examples of homogeneous hybridization assay methods (F luminophore, Q quencher, D donor, A acceptor). Thick lines represent DNA strands. Open circles on DNA strands indicate a SNP/mutation site for Molecular Beacon and insertion/deletion sites for dual FRET probe and dual FRET Molecular Beacon when these methods are applied to SNP/mutation typing or deletion/insertion detection. The solid circle on die strand indicates the complementary site.

Invader Assay. The Invader assay developed by Third Wave Technologies, Inc. (Madison, WI) is an attractive FRET-based technique to genotype SNPs without PCR amplification (Figure 7.5-5). Two oligonucleotides, a wild-type or variant signal probe plus an upstream Invader probe, are used in each reaction. These... 

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