Fluorescence resonance energy transfer

Fluorescence resonance energy transfer (FRET) occurs where two fluorophores are in close proximity, and one of the fluorophores (the donor) has an emission spectrum that overlaps the excitation spectrum of the other fluo-rophore (the acceptor). When a lone donor fluorophore is excited, light is produced with a characteristic emission spectrum. However, when the donor fluorophore is excited in close proximity to the acceptor fluorophore, energy is transferred to the acceptor fluorophore with the result that the intensity of emission from the donor is reduced (quenched), whereas that of the acceptor is increased (enhanced).

Although FRET enhancement can be used for SNP genotyping, two of the most popular homogenous assays in current use employ FRET quenching. The TaqMan assay, mentioned earlier, uses the intrinsic 5' nuclease activity of Taq DNA polymerase to generate a fluorescent signal from a short ASO probe.[12] Two ASO probes are required—one specific for each allele. Each probe contains a unique donor fluorophore and a common acceptor fluorophore, and is short enough for the donor to be quenched when the probe is intact (either hybridized or in solution). However, when the probe hybridizes to the PCR template, the 5' exonuclease activity of Taq DNA polymerase digests it, thus releasing the two fluorophores into solution and eliminating the quenching effect (Fig. 2a). The Molecular Beacon assay[13] involves the use of longer probes that have self-complementary ends labeled with the donor and acceptor fluorophores. Non-hybridized probes will self-anneal, bringing the donor and

Allelic Imbalance Taqman Assaywww.dekker.com.)"/>
Fig. 2 Principles of the two most popular fluorescence-based homogenous genotyping methods: (a) TaqMan assay; (b) Molecular Beacon assay. (View this art in color at www.dekker.com.)

acceptor fluorophores together, resulting in quenching. Where hybridization occurs, the donor and acceptor fluorophores are separated such that the quenching effect is eliminated and a fluorescent signal is produced. As with the TaqMan assay, two probes labeled with different donor fluorophores are required—one specific for each allele (Fig. 2b).

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

Get My Free Ebook

Post a comment