Glen Report 21.28: Deprotection - Volume 3 - Dye-Containing Oligonucleotides
Volume 3: Dyes - Deprotect to Completion
- Even with oligos containing sensitive dyes, the nucleobases must be fully deprotected for full functionality.
- Will the dye-labelled oligo survive my preferred deprotection scheme?
- If not, which deprotection scheme will fit best with my equipment and purification strategy?
In the previous two articles in this series on Deprotection, we focused on DNA and RNA deprotection. Our first priority in deprotection is to "Deprotect to Completion" by removing 100% of the protecting groups on the nucleobases, while following the mandate to "Do No Harm". Dyes tend to have the unfortunate properties of being quite sensitive to the basic conditions of oligonucleotide deprotection while being expensive. The "Do No Harm" stricture is doubly important when deprotecting dye-labelled oligonucleotides. To make matters worse, many dye labelled oligonucleotides also contain a quencher molecule that may also be base sensitive. This combination of properties is guaranteed to lead to confusion and possibly decomposed, worthless oligos may result if incompatible deprotection conditions are used.
In this article, we have generated a Table which we hope will remove some of the challenges from the deprotection of dye-labelled oligonucleotides. We have focused on a variety of methods for oligonucleotide deprotection:
- 30% NH4OH 17 hours at 55 °C; sufficient to deprotect all standard bases, A/C/G/T
- 30% NH4OH 17 hours at room temperature; sufficient to deprotect A, C and dmf-dG
- 30% NH4OH 2 hours at 65 °C; sufficient to deprotect A, C and dmf-dG
- 30% NH4OH 2 hours at room temperature; sufficient to deprotect only UltraMild monomers, Pac-dA, Ac-dC, ipr-Pac-dG when UltraMild Cap A is used.
- 50 mM Potassium Carbonate in Methanol for 4 hours at room temperature; sufficient to deprotect only UltraMild monomers, Pac-dA, Ac-dC, ipr-Pac-dG when UltraMild Cap A is used.
- Tert-Butylamine/water 1:3 (v/v) 6 hours at 60 °C; sufficient to deprotect A, C and dmf-dG.
- 30% Ammonium Hydroxide/40% Methylamine 1:1 (v/v) 10 minutes at 65 °C; sufficient to deprotect all standard bases, however, Ac-dC must be used.
The Table illustrates the conditions suitable for deprotecting oligos containing one or two of the dyes listed. We will continue to update this Table on our web site.
Note: JOE has not been tested with Condition F.
$ Denotes an acceptable, but not preferred method.
FAM | TET | HEX | JOE | TAMRA | Yakima Yellow |
Cy3/ DyLight 547 |
Cy5/ DyLight 647 |
Dabcyl | Eclipse | BHQ-1 | BHQ-2 | BHQ-3 | |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
FAM | A..G | A..G | B, D, E,G | A..E, G | E, F | A..D, F | B..E, G | D, E | A..E, G | A..G | $A, B..E, $F,G | A..E | $B, D |
TET | A..G | A..G | B, D, E, G | A..E, G | E, F | A..D, F | B..E, G | D, E | A..E, G | A..G | $A, B..E, $F,G | A..E | $B, D |
HEX | B, D, E, G | B, D, E, G | B, D, E, G | B, D, E, G | E | B, D | B, D, E, G | D, E | B, D, E, G | B, D, E, G | B, D, E, G | B, D, E | $B, D |
JOE | A..E, G | A..E, G | B, D, E, | A..E, G | E | A..D | B..E, G | D, E | A..E, G | A..E, G | $A, B..D | A..E | $B, D |
TAMRA | E, F | E, F | E | E | E, F | F | E | E | E | E, F | E, $F | E | Incompatible |
Yakima Yellow |
A..D, F | A..D, F | B, D | A..D | F | A..D, F | B, C, D | D | A..D | A..D, F | $A, B, C, D, $F | A..D | $B, D |
Cy3/ DyLight 547 |
B..E, G | B..E, G | B, D, E, G | B..E, G | E | B, C, D | B..E, G | D, E | B..E, G | B..E, G | B..E, G | B..E | $B, D |
Cy5/ DyLight 647 |
D, E | D, E | D, E | D, E | E | D | D, E | D, E | D, E | D, E | D, E | D, E | D |
Dabcyl | A..E, G | A..E, G | B, D, E, G | A..E, G | E | A..D | B..E, G | D, E | A..E, G | A..E, G | $A, B..E, G | A..E | $B, D |
Eclipse | A..G | A..G | B, D, E, G | A..E, G | E, F | A..D, F | B..E, G | D, E | A..E, G | A..G | $A, B..E, $F,G | A..E | $B, D |
BHQ-1 | $A, B..E, $F,G | $A, B..E, $F,G | B, D, E, G | $A, B, C, D | E, $F | $A, B, C, D, $F | B..E, G | D, E | $A, B..E, G | $A, B..E, $F,G | $A, B..E, $F,G | $A, B..E | $B, D |
BHQ-2 | A..E | A..E | B, D, E | A..E | E | A..D | B..E | D, E | A..E | A..E | $A, B..E | A..E | $B, D |
BHQ-3 | $B, D | $B, D | $B, D | $B, D | Incompatible | $B, D | $B, D | D | $B, D | $B, D | $B, D | $B, D | $B, D |
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- Glen Report 21.22: Purification of CleanAmpâ„¢ DNA Oligonucleotides (DMT-ON)
- Glen Report 21.23: A 3'-Cap for Improved Target Affinity and Specificity
- Glen Report 21.24: Metallobase Nucleic Acid Modification
- Glen Report 21.25: tC and tCo: New Tricyclic Fluorescent Cytidine Analogues with a very Bright Future
- Glen Report 21.26: Non-Aqueous Oxidation for PACE Chemistry
- Glen Report 21.27: New Product - Azobenzene Phosphoramidite for the Introduction of Photo-regulated Functions in DNA
- Glen Report 21.28: Deprotection - Volume 3 - Dye-Containing Oligonucleotides
- Glen Report 21.29: New Products - Glen UnySupportâ„¢ Frits
- Glen Report 21.210: New products: Glen-Pak™ DNA Cartridge 3g and DNA 30mg 96-Well Plates
- Glen Report 21.211: Technical Brief – Synthesis of Long Oligonucleotides