Fret Assays

A typical hybridization probe reaction allows two probes to interact to a common target sequence. For a typical reaction, probes are designed to bind to a common target within 1-3 nucleotides separating the two probes. The close proximity of the probes and associated dyes allows for a FRET reaction to occur. A donor fluorophore on the terminal end of one probe is excited by a light source and in turn transfers energy which excites the acceptor fluorophore on the terminal end of the second probe. The resulting fluorescence is proportional to the amount of amplified Target DNA

When designing FRET probes, the two most important details are to ensure that both probes bind to the intended target and at the proper location. Given a target (template) sequence, Visual OMP can design a probe in the most thermodynamically desirable location, taking into account mishybridization at any other location, probe lengths, binding Tm, template secondary structure, hairpin folding, probe dimers and checks using BLAST. A subsequent design of a second probe can then be designed at any distance away from the original probe, and from either 5’ or 3’ end to complete the FRET design.

Visual OMP enables users to input their respective flourophores from a list of commercially available modifications to get even more precise thermodynamics. Furthermore, if FRET probes are bound directly adjacent to each other, Visual OMP contains proprietary “coaxial stacking” parameters that add stability to oligos for being directly next to each other, a factor that could indicate the true accuracy of a design solution.

By performing a simulation and using our proprietary NPLEX solver, Visual OMP can determine the correct thermodynamics, concentration and percent bound of the template with a single probe vs. template with 2 probes bound vs. template with no probes bound. We can solve the multi-variable equations that are necessary to determine the concentrations of these species while they are competing with each other. Lastly, the Sequence View Editor, another innovation from DNA Software, allows users to see the binding of both probes at the same time, while in competition with the secondary structure of the original template.

For information on other types of FRET based assays such as Scorpions, TaqMan, or Beacons, please see the associated pages.

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