The synthesis of oligonucleotides requires the use of highly reactive chemicals including oxidizing reagents, strong acids, and after synthesis, a strong base. In addition to building the oligonucleotide, these reagents unfortunately also give rise to some side reactions, a few of which will be mentioned below.
Depurination during oligonucleotide synthesis is one problem caused by the acidic conditions used to remove the 5'-DMT group of the growing oligonucleotide. Depurination of N-benzoyl-Adenine from deoxyAdenosine derivatives is the major problem; with depurination of N-isobutyryl-Guanine from deoxyGuanosine derivatives at 10-fold lesser risk. 3' located purines in the oligonucleotide sequence are at a greater risk with a supportbound dABz group being especially labile because 5' located purines are more sensitive to the acid than internally located purines flanked by nucleotides. Furthermore, purines located in the 3' end of the oligonucleotide sequence are subject to more rounds of acid treatment. Oligonucleotides will undergo strand break at depurinated riboses during basic removal of protective groups.
Another problem is modification of N-2-isobutyryl-deoxyGuanosine to 2-6-diaminopurinedeoxynucleoside during addition, oxidation and capping. 2-6-DAP is an analogue of deoxyAdenosine and will give rise to dG to dA transitions.
Use of Oligonucleotides
Please be informed that we cannot guarantee that an oligonucleotide will always perform according to the user's intentions in biological experiments. Due to the synthesis chemistry, a crude oligonucleotide preparation is a mixture of oligonucleotides. This mixture contains the full length correct sequence oligonucleotide, different amounts of short species of correct partial sequence and also a fraction of oligonucleotides containing point mutations. Also, DNA polymerases in vitro and in vivo may give rise to mutations during polymerization. Therefore, positive clones should always be sequenced before further use. Furthermore, we have seen that some DNA sequences for some reason may be poisonous and, as such, incompatible with any given host.
However, we do guarantee that the oligonucleotide synthesis has the specified sequence, etc., as stated on the accompanying Certificate of Analysis.