Cy3 Internal

Click here for a list of fluorophores.

Cyanine 3 (Cy3) is a fluorescent dye that belongs to the Cyanine family of synthetic polymethine dyes. Cy3 is reactive, water-soluble, and has an absorbance maximum of 550 nm and an emission maximum of 570 nm. It is available as both a phosphoramidite and an NHS ester, and is used to fluorescently label oligonucleotides at either the 5' or 3' end, or internally. Cy3 plays a particularly important role in real-time PCR applications, being used as the reporter moiety in TaqMan probes (1), Scorpion primers (2) and Molecular Beacons (3). For such probes, Cy3 is most commonly paired with the dark quencher BHQ-2, as the two have excellent spectral overlap.

Cy3 can also be used to label DNA oligos for use as hybridization probes in other applications, such as Fluorescent In-Situ Hybridization (FISH). In 2010, Stoeckler and co-workers (4) reported that Cy3 double-labeling of FISH probes (at both ends) that were specific to ribsosomal RNA targets in microorganisms at least doubles FISH signal intensity without affecting specificity. This Double Labeling of Oligonucleotide Probes for Fluorescence In Situ Hybridization (DOPE-FISH 0 strategy may provide an effective solution to the problem of low signal intensity, which is commonly observed when using corresponding singly-labeled FISH probes for microbe identification. As an added benefit, Cy3-doubly labeled probes were shown to increase the in situ accessibility of rRNA targets sites in microbes, which allows for greater probe design flexibility.

Applied Biosystems Proprietary Dyes & Possible Substitutions
Fluorophore	Color	Absorbance max (nm)	Emission max (nm)
VIC	Pink Red	538	554
Cal Orange 560	Pink Red	537	558
HEX	Pink Red	535	556
NED	Red Orange	546	575
Cy3	Red Orange	550	570
PET	Red Orange	558	595
Cy3.5	Red	588	604
ROX	Red	575	602
Texas Red	Red	583	603

Click here for a list of fluorophores.

References
1. Livak, K.J., Flood, S.J.A., Marmaro, J., Giusti, W., Deetz, K. Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization.PCR Methods Appl. (1995), 4: 1-6.
2. Thelwell, N., Millington, S., Solinas, A., Booth, J., Brown, T. Mode of action and application of Scorpion primers to mutation detection. Nucleic Acids Res. (2000), 28: 3752-3761.
3. Tyagi, S., Kramer, F.R. Molecular beacons: probes that fluoresce upon hybridization. Nat. Biotechnol. (1996), 14: 303-308.
4. Stoecker, K., Dorninger, C., Daims, H., Wagner, M. Double Labeling of Oligonucleotide Probes for Fluorescence In Situ Hybridization (DOPE-FISH) Improves Signal Intensity and Increases rRNA Accessibility. Appl. Environ. Microb.. (2010), 76: 922-926.
- Cy3 Internal