6022 6-HEX phosphoramidite [6-HEX 亚磷酰胺]

分子式：C46H52N3O10Cl6P；分子量：1050.62；用于标记Oligo，溶于DMF及DMSO；−20°C干燥避光保存

5’-Labeling of Oligonucleotides Using Dye Phosphoramidites

Because conventional automated synthesis proceeds from 3' to 5', the 5'-terminus is clearly a good choice for modifying oligonucleotides to make the Taqman Probes. As reported in a number of publications, the ability to attach a suitable molecule to the 5'-terminus for use as a label play a critical role in the continuing development of non-radioactive probes and in DNA sequencing and amplification. A general approach to the modification of the 5'-terminus is to use reagents that would couple to the 5'-hydroxyl of an oligonucleotide. Dye phosphoramidite reagents have been readily adapted for use in automated synthesizers with little or no modification to existing protocols. These reagents are well compatible with automated DNA synthesizers. In general, dye-labeled oligonucleotides can be deprotected at room temperature in concentrated ammonium hydroxide for a minimum of 24 hours, or shorter time that is appropriated for the protecting groups on the monomers being used. FAM, JOE, Dabcyl, TQ or BHQ-labeled oligos can be heated to 55°C in ammonium hydroxide for extended periods of time. However, TET and Cy3 labeled oligos are less stable and will tolerate only a few hours at 55°C. HEX and Cy5 labeled oligonucleotides must be deprotected at room temperature and the residual ammonia should be removed immediay after deprotection.

Fluorescein Labeling: These Fluorescein-based phosphoramidites that contains no 4,4’-dimethoxytrityl （DMT） group and can be added only once at the 5’-terminus, thereby terminating synthesis. These fluorescein products include FAM, TET and HEX. FAM phosphoramidite is designed to produce the same fluorescein-type structure as had been previously prepared using fluorescein isothiocyanate （FITC）. The TET and HEX phosphoramidites are designed to take advantage of the multicolor detection capability of modern DNA sequencers and genetic analyzers.

Rhodamine Labeling: The light-absorbing properties of TAMRA, and spectral overlap with several commonly used fluorophores - including FAM, HEX, TET and JOE, make it useful as a quencher for the duallabeled oligo probes. However, its intrinsic fluorescence contributes to the background signal, potentially reducing the sensitivity of assays based on TAMRA. Despite these limitations, TAMRA has been used extensively in the design of probe-based assays, perhaps most notably for Real-Time PCR. Oligonucleotides can be labeled with TAMRA using two distinct methodologies. Under standard deprotection conditions, TAMRA is not sufficiently stable. It degrades in the presence of ammonium hydroxide. If standard deprotection is required, the oligonucleotide is normally synthesized with an amino group at either the 3’-, or 5’-end and labeled with TAMRA post-synthetically using TAMRA, SE. Oligonucleotides synthesized using UltraMILD monomers can also be labeled directly with TAMRA at the 3’-end using 3’-TAMRA CPG support. Subsequent deprotection of the oligo using potassium carbonate in methanol adequay removes the base protecting groups.

Quencher Group Labeling: Quencher dyes are optimized to maximize FRET performance through enhancing donor fluorescence intensity. Such as Dabcyl, BHQ, TQ, EDANS, FAM, TAMRA, ROX, Cy3 and Cy5 have been widely used to develop a variety of FRET probes.

The following procedure to label oligonucleotides with 6-FAM phosphoramidite is Fanbo’s protocol in house, customers should optimize the protocol based on their own condition.

6-FAM phosphoramidite

CAS#：204697-37-0

Cat#：6020

Molecular Formula：C₄₆H₅₈N₃O₁₀P

Molecular Weight：843.94

Incorporation of 6-FAM phosphoramidite into Oligo

1.  Use Dry MeCN （H₂O content< 30ppm） to dissolve the FAM phosphoramidite. It is important to maintain anhydrous conditions when dissolving the FAM phosphoramidite in anhydrous MeCN.

2.  For use on PE™ 8900 instruments, add 2ml MeCN to 0.1g FAM phosphoramidite to obtain a concentration of 50mg/ml. For use on PE 390 series instruments, add 1.2ml MeCN to 0.1g FAM phosphoramidite to prepare a 0.1M solution.

3.  Gently swirl the vial until the powder is compley dissolved.

4.  Once the FAM phosphoramidite has been dissolved and placed on your instrument, it should be used within 3 days.

5.  Attach the dissolved phosphoramidite to the appropriate position on the synthesizer. Ensure that the delivery line to the synthesis chamber is sufficiently primed.

6.  Enter the sequence of the oligonucleotide you wish to synthesize with FAM phosphoramidite at the 5’-end. A minimal coupling time of 3 minutes is recommended for FAM phosphoramidite.

7.  Proceed as you would with a standard DNA oligonucleotide synthesis. Note this FAM phosphoramidite does not contain a DMT group. Oligonucleotides do not need to be detritylated at the end of the synthesis. Note that this FAM phosphoramidite will terminate the synthesis and can only be employed in the last coupling step on the 5’-terminus.

8.  Cleave and deprotect the oligonucleotide with ammonia at 55°C for 8 hours with standard protected nucleobases, or, if TAC-protected phosphoramidites are used, at 55°C for 15 minutes. The FAM- moiety is stable under these conditions.

9.  The oligonucleotide is now ready for further processing, such as desalting or purification with RP-HPLC, AX-HPLC or gel base method methods. The FAM label allows the purified fraction to be easily detected during collection.

10.  Oligonucleotides labeled with dye should be stored in the dark.

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