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:
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 |