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).
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.
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.
3. Tyagi, S., Kramer, F.R. Molecular beacons: probes that fluoresce upon hybridization. Nat. Biotechnol.
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.
6-FAM dT Fluorescent Dye Oligonucleotide Labeling
-20°C To -70°C
Oligonucleotides are stable in solution at 4°C for up to 2 weeks. Properly reconstituted material stored at -20°C should be stable for at least 6 months. Dried DNA (when kept at -20°C) in a nuclease-free environment should be stable for years.
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