6-FAM Fluorescent Dye Internal Oligonucleotide Labeling

6-carboxyfluorescein (6-FAM), representing the most commonly used fluorescent dye for labeling oligonucleotides, is reactive and water-soluble (absorbance maximum of 492 nm; emission maximum of 517 nm).  6-FAM especially useful in real-time PCR, being used as a reporter moiety in TaqMan probes, Scorpion primers, and Molecular Beacons (1,2,3).   For the above applications, 6-FAM is commonly paired with the dark quencher BHQ-1, as they exhibit excellent spectral overlap.   6-FAM-labeled primers were used in bacterial SNP genotyping via allele-specific real-time PCR (4).  6-FAM labeled oligonucleotides (DNA) as hybridization probes are useful for various in vivo and in vitro research/diagnostic applications, and for structure-function studies of DNA, RNA, and protein-oligonucleotide complexes.   Oligonucleotides labeled with 6-FAM at 5’-end can be used to generate fluorescently-labeled PCR products, as primers for DNA sequencing, or for genetic analysis (AFLP or microsatellite).

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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. Huygens, F., Inman-Bamber, J., Nimmo-G.R., Munckhof, W., Schooneveldt, J., Harrison, B., McMahon, J.A., Giffard, P.M. Staphylococcus aureus Genotyping Using Novel Real-Time PCR Formats. J. Clin. Microbiol. (2006), 44: 3712-3718.