FRET stands for fluorescence resonance electron transfer. This phenomenon is observed when two dyes are placed within proper distance of each other (one full turn of DNA is an excellent length). If the first dye is excited and emits photons, and the second dye absorbs that wavelength, the only observed emitted wavelength will be that of the second dye. This ability to see another wavelength when two dyes come into proximity of each other has a number of useful applications for diagnostic assays.

At most, we recommend one conjugate every 5-10 bases. Theoretically, we can place a conjugate at every location and at the 3’ and 5’ ends. Unfortunately, it is very difficult to synthesize these types of compounds and just as difficult to analyze for labeling efficiency. In many cases, the resulting products are essentially insoluble. In other cases, such as fluorescein, the dye actually quenches itself if too many are present, becoming almost non-fluorescent. The length is also limited.

It is possible to place up to six different dyes on one oligonucleotide, but that can only be done with a very specific sequence of dyes and is of no practical use. We routinely place two dyes on an oligonucleotide, and can place up to four with some degree of flexibility in dye choice using a combination of synthesis reagents and an orthogonal linker scheme. However, any combination above two gets more problematic to synthesize and purify, which will affect purity and yield.  Please contact us to discuss your specific needs.

The answer is complicated because there are many ways to place different conjugations on an oligonucleotide, but we are limited by what is available for conjugation and by compatibility issues. The easiest way is if one or both of the conjugates are available as a phosphoramidite and/or support bound reagents. If at least one reagent is available, the other conjugate can often be attached through a primary amine at the other end of the oligonucleotide. Chemical compatibility is avoided in this case. If neither conjugate is available as a phosphoramidite or support bound reagent, then we often will use an orthogonal linker scheme incorporating both a thiol linker and an amino linker. This requires that one of the conjugates is available as a maleimide, which is thiol specific under neutral pH conditions. Most of all the commonly used dyes are available in both the succinimidyl and maleimidyl ester forms. The most common, such as fluorescein, TET, HEX, TAMRA, Cyanine 5 and Cyanine 3 are also available as phosphoramidites or support bound reagents. We will help you design your specific oligonucleotide when you place your order.

Spectra data is listed for each dye on our website and in our catalog. These values are provided by the manufacturer of the fluorescent dye and are generally calculated from the free dye not attached to an oligonucleotide. As a general rule, these values work fine for the common oligonucleotide user as there is little change, if any, when the dye is attached to an oligonucleotide. The particular base composition of an oligonucleotide can play a role as can pH in many cases, particularly with fluorescein and related dyes.

Yes. Tell us the dye or modification you are looking to use; we will find out if it is commercially available. If it is not, there is a possibility we can make it in-house. If neither of these is an option we will do our best to see if there is another dye or modification that can give you similar results. If any of these options work for you, we will then send you a quotation.

The original supply of reagent was finally exhausted, and the cost of replacement far outweighed the potential sales. We had to make the unfortunate decision to relinquish our license.

A quencher is very efficient at absorbing certain wavelengths. When near a dye that emits at the absorbed wavelength, the light is “quenched”, and no longer visible. Quenchers are very similar in structure to dyes. The difference is they emit in undetectable ranges, or in undetectable amounts. The ability to quench is a function of distance from the dye in most cases. Molecular beacons are effective in that the quencher actually comes in contact with the dye. Different quenchers are best for different dyes.

This is a difficult question since so much depends on your specific target sequence and application. A good set of rules for the design of molecular beacons can be found on www.molecular-beacons.org. In general, the most successful compounds are 30-40 nucleotides in length, with the stem comprising the last 6 bases on either terminus. Another general rule is that the melting temperature of the stem should be about 10°C lower than that of the target/probe section of the molecular beacon. This is where the trial and error comes in as the design tools are only predictive of nature.

Both 6-FAM (6-carboxyfluorescein, single isomer) and FITC (fluorescein-5,6-isothiocyanate, mixed isomers) are forms of the fluorescent dye fluorescein. FITC is a particular form of reactive species, the isothiocyanate, of the dye. It yields a urea linkage upon reaction with a primary amine. 6-FAM is the preferred reagent for labeling an oligonucleotide. It results in an amide bond when reacted with a primary amine. The chemistry is more robust and better yielding. Furthermore, it has been shown that 6-FAM is less susceptible to photobleaching. 6-FAM and FITC conjugated oligonucleotides have similar spectral properties otherwise.

Yes. We use the succinimidyl ester version of the dye and conjugate it to a selective placement linker. We then post-synthetically conjugate the dye to the oligonucleotide. Other options include using a dT-FAM or dT-TAMRA. These dyes are already conjugated to the dT-C6 amino linker.