3'-Dabcyl CPG Column
3'-Dabcyl CPG Column
Key featuresShow Hide
- Used to add a yellow-green fluorescent quencher to the 3' end of an oligonucleotide.
- Emission range at 400-550 nm (absorption maximum, 471 nm). Alternative to BHQ-1 in certain applications.
- 1000 Å CPG suitable for highly modified oligonucleotides (> 20mers).
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Dabcyl is a non-fluorescent molecule, (so-called dark quencher) efficiently quenching various fluorescent dyes. It has been widely used as a quencher in diagnostic probes such as Molecular Beacons.(1) A Molecular Beacon is a hybridisation probe consisting of a fluorophore, a quencher and a defined section of the oligonucleotide sequence complementary to that of the target nucleic acid. In the inactive state, when the probe is not hybridised to its target sequence, the fluorescence energy of the fluorophore is transferred to the quencher by a process of collisional quenching. For light energy transfer to take place efficiently, both fluorophore and quencher have to be in close proximity. This requirement is accounted for in the design of Molecular Beacons in that the two parts of the stem hybridise to hold the F/Q pair in close proximity.
Hybridisation of the Molecular Beacon probe to its target sequence results in the separation of the stem and hence the F/Q pair, resulting in fluorescence.
We offer a variety of CPG and phosphoramidite Dabcyl products for myriad applications. Since Dabcyl is stable to oligo synthesis it can be incorporated at any point in the sequence. 5’ modification is used in TwistAmp fpg probes; 3’ modification in Taqman probes, duplex Scorpions and Molecular Beacons; and internal modification has been utilised in Scorpion Primers. Deprotection of the oligo is dependent on the F/Q pair but in general is as per unmodified oligos.
Dabcyl’s absorption properties limit the range of dyes it can quench to those emitting at 400-550 nm such fluorescein and JOE (absorption maximum, 471 nm), but tends to overlap poorly with fluorophores emitting above 480 nm. However, when used in Molecular Beacons, the fluorophore and Dabcyl are brought close enough to allow a slightly broader spectrum of dyes to be quenched, thereby increasing the versatility of the Dabcyl molecule.
Although Dabcyl was originally the quencher of choice, for the most part this has been superceded by Black Hole Quenchers (BHQs) for newer applications.
- Molecular beacons: probes that fluoresce upon hybridisation, S. Tyagi and F.R. Kramer, Nature Biotechnology, 14, 303-308, 1996.
Physical & Dilution Data
Dilution volumes (in ml) are for 0.1M solutions in 10% THF in dry acetonitrile (LK4059) for LK2144, and 0.1M solutions in dry acetonitrile (LK4050) for LK2085. Adjust accordingly for other concentrations. For µmol pack sizes, products should be diluted as 100µmol/ml to achieve 0.1M, regardless of molecular weight.
Today, the most commonly used dark quenchers are all azo based. It must be noted that as a result it is not uncommon to observe fragmentation during mass spectrometry analysis by MALDI-TOF. Dabcyl and DDQ-1 will show a fragment of M-134, BHQ™-1 shows M-298 and BHQ™-2 shows M-300.
Dabcyl-dT-CE Phosphoramidite (LK2144) is dissolved in 10% THF in acetonitrile, 5’-Dabcyl-CE Phosphoramidite (LK2085) in anhydrous acetonitrile.
A 6min coupling time is recommended for the phosphoramidites. 3’-Dabcyl CPG (LK2374) and 3'-Dabsyl-CPG (LK2426) are used as per standard nucleoside supports. However, since non-nucleosidic modifications detritylate at a slower rate it is recommended to carry out an initial additional detritylation. In this case it is important not to carry out any additional capping.
Standard oligonucleotide deprotection conditions can be applied when deprotecting an oligo containing this modification; typically AMA, 55oC, 35min.
Storage & Stability
All compounds are stored in a freezer at -10 to -30°C. Stability of the phosphoramidites in solution is 2-3 days.