LC Red 640 NHS

LC Red 640 modification is a post synthesis conjugation to a primary amino group thus an additional modification with an amino group is required. A C6 or C12 amino group can be placed at the 5' or for the 3' end a C3 or C7 amino and for internal positions an amino modified base is used, e.g Amino dT C6.

LC Red 640 NHS Ester is a fluorescent dye for labeling oligonucleotide hybridization probes used in LightCycler qPCR assays. LC Red 640 has an absorbance maximum of 625 nm and an emission maximum of 640 nm. LightCycler (“adjacent probe”) assays require two single-stranded hybridization probes which bind to adjacent sites on a target strand. One probe is 3’-end-labeled with a donor fluorophore (typically fluorescein), while the other probe is 5’-end labeled with LC Red 640 as the acceptor fluorophore (and blocked at its 3’-end with phosphate). The distance between the 3’ and 5’-ends of the respective probes, when hybridized to the target, is carefully chosen to ensure efficient fluorescence resonance energy transfer (FRET) between donor and acceptor fluorophores. When not hybridized to the target, that is, when they are “free-floating” in solution, no FRET should occur, and only the fluorescence of the donor fluorophore should be present. However, when both probes are hybridized to the target, FRET should occur, resulting in a decrease in donor fluorescence and increase in acceptor fluorescence (1).

LightCycler assay systems have been developed for SNP detection (2), allelic discrimination (3), gene copy determination (4), pathogen detection (5), viral load quantification (6), and gene expression analysis (7). Additional red dyes suitable for use as acceptor fluorophores, namely LC Red 610 and Cy5.5, permit LightCycler assays to be in multiplex format.

References
1. Wittwer, C.T., Herrmann, M.G., Moss, A.A., Rasmussen, R.P. Continuous fluorescence monitoring of rapid cycle DNA amplification. Biotechniques (1997), 22: 130-131.
2. Hiratsuka, M., Narahara, K., Kishikawa, Y., Ismail, H.S., Endo, N., Agatsuma, Y., Matsuura, M., Inoue, T., Tomioka, Y., Mizugaki, M. A simultaneous LightCycler detection assay for five genetic polymorphisms influencing drug sensitivity. Clin. Biochem. (2002), 35:35-40.
3. Mangasser-Stephan, K., Tag, C., Reiser, A., Gressner, A.M. Rapid Genotyping of Hemochromatosis Gene Mutations on the LightCycler with Fluorescent Hybridization Probes. Clin. Chem. (1999), 45: 1875-1878.
4. Ruiz-Ponte, C., Loidi, L., Vega, A., Carracedo, A., Barros, F. Rapid Real-Time Fluorescent PCR Gene Dosage Test for the Diagnosis of DNA Duplications and Deletions. Clin. Chem. (2000), 46: 1574-1582.
5. Wellinghausen, N., Wirths, B., Franz, A.R., Karolyi, L., Marre, R., Reischl, U. Algorithm for the identification of bacterial pathogens in positive blood cultures by real-time LightCycler polymerase chain reaction (PCR) with sequence-specific probes. Diagn. Microbiol. Infect. Dis. (2004), 48: 229-241.
6. Gulley, M.L., Fan, H., Elmore, S.H. Validation of Roche LightCycler Epstein-Barr virus quantification reagents in a clinical laboratory setting. J. Mol. Diagn. (2006), 8: 589-597.
7. Frade, J.P., Warnock, D.W., Arthington-Skaggs, B.A. Rapid Quantification of Drug Resistance Gene Expression in Candida albicans by Reverse Transcriptase LightCycler PCR and Fluorescent Probe Hybridization. J. Clin. Microbiol. (2004), 42: 2085-2093.
- LC Red 640 NHS