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Chemical
Composition and Purpose of Proligo Reagents' Liquid Reagents During the
Synthesis Cycle
Deblock
solution,
containing trichloroacetic acid (TCA) or dichloroacetic acid
(DCA) in dichloromethane, removes the dimethoxytrityl (DMT) protecting
group from the 5' hydroxyl moiety of nucleotides already incorporated into
the growing nucleic acid, prior to the addition of the next
phosphoramidite. Removal of the DMT allows the unprotected 5' hydroxyl
moiety to react with a new phosphoramidite in a subsequent extension
reaction.
Wash
solution,
containing anhydrous acetonitrile (water content <
30ppm), is used as the overall wash solvent following each step in the
synthesis cycle.
Activator
solution,
containing 4,5-dicyanoimidazole (DCI) in acetonitrile, is
typically added simultaneously with a phosphoramidite during the extension
step. The activator reacts with the trivalent phosphorous of the
phosphoramidite to form a reactive intermediate. This intermediate then
forms an internucleotide phosphite with the detritylated 5' hydroxyl group
of the growing oligonucleotide chain.
Activator 42™
- Among the highest activation efficiency in the industry compared to
other activators
- Promotes ultra-fast DNA couplings (coupling time less than 15
seconds)
- Promotes rapid RNA couplings (coupling time 6 minutes)
- Excellent solubility in acetonitrile (greater than 0.9M)
- Not hygroscopic
- Safe to handle (no explosive properties)
- Very high overall yield in DNA- and RNA-synthesis
Cap
A solution,
containing acetic anhydride and tetrahydrofuran (THF), is
used after the phosphoramidite reaction/coupling step. This solution
aborts chains bearing unreacted 5' hydroxyl groups due to failure of
reaction with activated phosphoramidite (typically, 1 to 2% of growing
oligonucleotide chains in each synthesis cycle). These unreacted 5'
hydroxyl groups are capped with an acetyl group and thus rendered
unreactive for subsequent synthesis steps. For synthesis with Proligo
Reagents' TAC-protected phosphoramidites, Fast Deprotection CAP A solution
must be employed instead of Cap A solution.
Fast
Deprotection Cap A solution,
must be used for synthesis with Proligo
Reagents' TAC-protected phosphoramidites. Fast Deprotection Cap A
solution, containing tert-butylphenoxyacetyl acetic anhydride (tac2
O) in tetrahydrofuran, is used in place of Cap A solution to ensure that
the displacement of tert-butylphenoxyacetyl (TAC) does not occur.
Cap
B solution,
containing tetrahydrofuran (THF), pyridine and
1-methylimidazole, is mixed in-situ with Cap A solution during the capping
(acetylation) reaction. The pyridine maintains a pH basic enough to
promote the acetylation reaction and the 1-methylimidazole, together with
the acetic anhydride, makes an excellent acetylating agent.
Oxidizer
solution,
containing tetrahydrofuran (THF), pyridine, iodine and
water, is used to oxidize the chemically unstable trivalent phosphorous
triester linkage after the coupling reaction to the stable pentavalent
phosphate triester linkage of natural nucleic acids. Here, iodine
functions as a mild oxidant and water functions as an oxygen donor.
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