Dear Aaron,
There are two concerns raised in you message, depurination and n+1 products,
but unlike you, I do not think they have the same origin.
Depurination will invariably result in backbone cleavage on deprotection,
leading to truncated oligonucleotides. Therefore, I would not try to
explain the n+1 shoulder on your CE with depurination. Our own experience
shows that when very long oligos are used for cloning, the n+1 products
detected by sequencing are usually G duplications, and the reasons for those
are not well understood (or it is me the ignorant). The n+1 products
detectable on HPLC are thought to be most often caused by chain branching at
G.
Because G branching is not caused by TCA, the practical question is whether
depurination, which is indeed linked to TCA, should be avoided by replacing
TCA with DCA or decreasing the time for TCA exposure (deblocking). I would
strongly recommend against, because truncated oligos (resulting from
depurination) do not seem to be your problem, and because by milder
deblocking (DCA, or shorter time for TCA) you risk to increase the n-1
products. One of the preventable reasons for n-1 products is the incomplete
deblocking, and the frequency of the n-1 products is an order of magnitude
higher than n+1 products. Therefore, I would stick to the TCA, and even
increase the time for treatment with TCA, if n-1 products come to haunt me
(but again, others on this list will say this is ludicrous).
BTW, could it be that the n+1 product on your CE is in fact an n+DMT product
(DMT's m.w. being equal to one nucleotide), caused by incomplete deblocking
because of your fear of TCA? (just kidding)
Best regards,
victor
www.alphadna.com
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