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Many oligonucleotide synthesis facilities struggle to produce good quality long oligos, and researchers often mistakenly believe that this is due to deficiencies in the purification protocol. This is usually not the case, a flawed synthesis cannot be rescued in the purification step. It is an inescapable fact that the longer the oligonucleotide, the more difficult the synthesis. Failure sequences build up with increasing oligo length, and long oligos contain impurities that are, by definition, similar in properties to the desired sequence. If failure sequences are allowed to accumulate it is impossible to obtain pure oligos because the capping step in oligonucleotide synthesis cannot be 100% efficient. This results in a significant (n-1) impurity which is a mixture of deletion mutations − potentially disasterous in biological applications.
The synthesis of long oligonucleotides is demanding; it requires the very best in equipment, reagents, protocols and expertise. We have all of these at ATDBio.
By optimizing the synthesis methodology we can produce long oligos of sufficient quality and quantity to give the purification the best chance of succeeding. We then use our knowledge of oligo purification methods to produce the purest oligo possible.
After synthesis and purification, we do rigorous and exhaustive analysis of the oligos, using mass spectrometry, capillary electrophoresis and analytical HPLC. Denaturing capillary electrophoresis is particularly relevant for long oligonucleotides as they are particularly susceptible to secondary structure formation.