I have sequenced a clone I have prepared with your primer and the sequence for the primer region was different from the one I have ordered. Why?
First of all, we strongly recommend our customers to sequence more than one clone and compare the sequencing results. In most cases, sequencing at least 3 clones is sufficient to find the clone with the desired sequence. This is also due to circumstances out of oligo manufacturers´ control, like sequence errors generated by enzymes like Taq polymerase used in downstream experiments. PCR-cloned sequences may contain errors due to the inherent "infidelity" of any kind of available polymerases. Taq may have error rates as high as 0.25%. If Taq was not used, the difference could be due to a recombinant vector or the host cell system "self-correcting" errors.
The problem can also be due to the primers. Base insertions are attributed to a small amount of detritylated amidite present during coupling, while deletions are probably due to failure sequences that did not get capped and were subsequently extended.
However, a better explanation for the observation of altered sequences is the incomplete deprotection of the oligo. If an oligo still bears a protecting group in one or more positions, this will be transferred to the subsequent PCR product, which is then transformed into E. coli. Here, the host mismatch repair system will likely attempt to correct the corresponding anomaly with a base, which might be the wrong one. The most likely culprit for incomplete deprotection is the isobutyryl protected dGs. These are the most difficult protection groups to remove.
In general, the longer the oligo, the greater the probability of side reactions during oligo synthesis, along with higher chances to incur into incomplete deprotection. Potential sources of side reactions causing failure products are depurination (which mainly affects the base A) and formation of secondary structures due to the oligos’ sequence. There is no way to completely exclude these effects! However, metabion tries to minimise these failures by continuously optimising synthesis as well as purification protocols!