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Coupling efficiency is important because the effects are cumulative during DNA synthesis. The Table below shows the effect of a 1% difference in coupling efficiency and how this influences the amount of full-length product, following the synthesis of oligos of different length. Considering a relatively short oligo of 20 bases, a 1% difference in coupling efficiency can result in a 15% difference, in terms of full-length final product.
For the 0.2 µmol synthesis scale, monomer coupling is done at a 40-50-fold excess. Doing so for larger scale synthesis (such as for a 1.0 µmol scale) would be cost-prohibitive. Large-scale syntheses are done only at 10-fold mole excess of amidites. To increase the final yields of these large-scale synthesis, the coupling times are extended, so to increase coupling efficiencies.
custom oligo synthesis, oligonucleotides ..
For oligos of standard length (≤ 33 nucleotides) we guarantee a certain yield of DNA (OD260 applying to a 20-mer) to be delivered according to the respective scale of synthesis and mode of purification.
Oligos are made using a DNA synthesizer, which is basically a computer-controlled reagent delivery system. The first base is attached to a solid support, usually a glass or polystyrene bead, which is designed to anchor the growing DNA chain in the reaction column. DNA synthesis consists of a series of chemical reactions.
Custom DNA and RNA Oligos & qPCR Probes
The trityl-group is colorless when attached to a DNA base but it gives a characteristic orange color once removed. The intensity of this color can be measured by UV spectrophotometry and it is directly related to the number of trityl molecules present. Following the first coupling step, the amount of trityl released during deblocking is directly proportional to the amount of full-length oligo synthesized in the previous cycle. When the trityl is cleaved during the deblocking step, the resulting trityl cation is orange in color. The intensity of this color can be measure by UV spectrophotometry. By comparing the intensities of the trityl cation produced after the first and last coupling steps, one can calculate the average successful base coupling per cycle and hence the coupling efficiency.
DNA synthesis is a complicated process, which has improved significantly over the last years. Despite these improvements, all manufacturers have an inherent failure rate. We are constantly developing our processes and systems to minimize these losses; however, it is inevitable that we will occasionally have to re-synthesize some oligos. Please note that metabion performs strict quality controls on each and every oligo synthesized. If an oligo does not pass our quality tests, it will be resynthesized.
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