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  6. 3'-TAMRA CPG

3'-TAMRA CPG

3'-TAMRA CPG

3'-TAMRA CPG

A CPG used for 3'-TAMRA (rhodamine) labelling of an oligonucleotide

Key features

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  • Used to add a broad range green-blue quencher to the 3' end of an oligonucleotide.
  • Will quench FAM, HEX, TET and JOE; perfect for dual-labelled probes and probe-based assays.
  • Also has fluorophore properties, emission maximal at 580 nm.
  • 1000 Å CPG suitable for highly modified oligonucleotides (> 20mers).
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Product information

TAMRA (carboxytetramethylrhodamine) exists as 5- and 6-derivatives, or as a mixture of both isomers. It is the most commonly used rhodamine dye in oligonucleotide based applications. The fluorescent properties of TAMRA are sometimes used in non-radioactive sequencing, and in situ hybridisation studies, however TAMRA is more often as a quencher in Real-Time PCR applications based on FRET.

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 design of dual-labelled probes. The usefulness of TAMRA is, however, limited because of its broad emission spectrum, which reduces its capabilities in multiplexing. 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 in Taqman probes for Real-Time PCR.

TAMRA is not sufficiently stable to strong bases; the molecule degrades in the presence of ammonium hydroxide, hence TAMRA-labelled oligos must be synthesised using mild deprotection monomers. Subsequent deprotection of the oligo is achieved with t-butylamine/methanol/ water (1:1:2) for 2.5h at 70˚C. Although there is still a small amount of TAMRA degradation, this is easily removed during purification. Alternatively, amino-modified oligos can be post-synthetically labelled using a suitable TAMRA NHS ester.

We provide a number of solid support and phosphoramidite options for direct TAMRA labelling internally, or at the 3’ or 5’ end of oligonucleotides.

Applicable Products

LK0251 TAMRA NHS Ester in DMSO
LK2143 TAMRA-dT-CE Phosphoramidite
LK2434 3'-TAMRA CPG S
LK2435 3'-TAMRA CPG L

Physical & Dilution Data

Dilution volumes (in ml) are for 0.1M solutions in 10% THF in anhydrous acetonitrile. Adjust accordingly for other concentrations. For µmol pack sizes, products should be diluted as 100µmol/ml to achieve 0.1M, regardless of molecular weight.

Item

Mol. Formula

Mol. Wt.

Unit Wt.

250mg

500mg

1g
LK0251 C29H25N3O7 527.53 412.44 - - -
LK2143 C73H83N8O13P 1311.48 870.85 1.91 3.81 7.62
LK2434/5 - - 623.60 - - -

Synthesis Planning

TAMRA-labelled oligonucleotides require milder deprotection conditions: use tbutylamine/methanol/water (1:1:2).1 Therefore, use Ac-dC and dmf-dG when synthesising the oligo being labelled. Standard ammonium hydroxide deprotection significantly degrades TAMRA. Post-synthetic labelling using TAMRA NHS Ester (LK0251) is discussed below.

Dissolution

TAMRA-dT-CE Phosphoramidite (LK2143) is dissolved in 10% THF in anhydrous acetonitrile (LK4059) to standard concentrations. Allow 15min for the product to completely dissolve.

Coupling

LK2143 – A 6min coupling time is recommended.

Cleavage & Deprotection

LK2143 – Deprotection is carried out using tbutylamine/methanol/water (1:1:2) for 2.5h at 70˚C.

LK2434/5 – Typical protocol is as follows:

  1. After the oligo has been synthesised, remove the support from the column and add 600μl of tbutylamine/methanol/water (1:1:2) solution.
  2. Heat at 70˚C for 2.5h..
  3. Pass the sample down a G25 column equilibrated with water. This removes the deprotection solution.
  4. Sample is now ready for purification.

Poly-Pak™ Cartridge Purification2

Note that the TAMRA label degrades during deprotection and these degraded oligos are not removed during Poly-Pak™ purification.

Typical protocol is as follows:

Cartridge Preparation

  1. Connect a syringe to the female luer of the Poly-Pak™ II cartridge and terminate the male luer in a waste vessel.
  2. Flush the cartridge with 4ml acetonitrile followed by 4ml 2M TEAA.

Sample Preparation

  1. Following synthesis, deprotect the oligo as outlined above.

Purification

  1. Add 1 volume 0.1M TEAA with 8% acetonitrile followed by 8 volumes deionised water to the cartridge.
  2. Load the sample solution onto the cartridge. Reload if necessary after addition of a few drops of 2M TEAA which increases the affinity of the oligo for the support.
  3. Flush the cartridge with 4ml 2% TFA; do this whether the oligo is DMT ON or OFF.
  4. Rinse the cartridge with 10ml 1:3 – 1:4 solution of acetonitrile:2% TFA. Note: the percentage of acetonitrile used depends upon the length and sequence of the oligo. Using a 1:4 ratio is appropriate for oligos approximately 20 nucleotides long.
  5. Flush the cartridge with 4ml distilled water.
  6. Rinse the cartridge with 10ml of 0.1M TEAA containing 12% acetonitrile. This is to remove non-labelled oligos. If significant TAMRA is seen coming off the cartridge, stop the rinse.
  7. Elute the product with 2ml of 50% acetonitrile in water.

Post-Synthesis Labelling Protocol

As noted above, TAMRA-labelled oligos will not survive standard deprotection conditions. Therefore, if such conditions are necessary, LK0251 can be used to label the oligonucleotide post-synthetically. This is provided as a 0.17M solution in DMSO. A synthesised amino-modified oligonucleotide is then conjugated to TAMRA using the NHS ester in sodium carbonate/bicarbonate buffer (0.1M, pH=9). A typical protocol for the conjugation of an amino-modified oligo (synthesised at 0.2μmol scale) is as follows:

  1. Dissolve the amino-modified oligo in 125μl of conjugation buffer.
  2. Add 6μl of TAMRA/DMSO solution (ca. 5-fold excess).
  3. Vortex the mixture and incubate at 37°C in the dark overnight.
  4. Separate the oligo-TAMRA conjugate from salts and free TAMRA by size-exclusion chromatography on a NAP-10 column or equivalent.
  5. To do this, equilibrate the NAP column with 0.1M TEAA.
  6. Load the reaction mixture onto the column and let this flow into the column.
  7. Add 0.5ml TEAA buffer and also let this flow into the column.
  8. Elute the conjugate with <1.5ml TEAA buffer.
  9. Collect the conjugate and reduce to a suitable volume for HPLC purification.

Spectral Data

 

Absorbance Max./nm

Emission Max./nm

Colour
TAMRA 565 580 Pink

Storage & Stability

All products are stored dry in a freezer at –10 to –30°C, protected from light. Labelled oligos are stored in the dark. LK2143 is stable in solution for 24h.

References

  1. Automated synthesis of double dye-labelled oligonucleotides using tetramethylrhodamine (TAMRA) solid supports, B. Mullah and A. Andrus, Tetrahedron Lett., 38, 5751-5754, 1997.
  2. Poly-Pak™ Purification of 3’-TAMRA Labelled Oligos, v1.0, 5/99, Glen Research. Poly-Pak™ is a trademark of Glen Research.

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