During DNA synthesis, each new synthesis cycle leaves so called truncated sequences. No process successfully synthesises 100% of the material, at best perhaps 99%. Each additional step in the process increases the number of truncated and non-elongated oligonucleotide chains. By way of example, consider the 10mer. With a synthesis or coupling efficiency of 99%, then complete chains represent (99%)9 = 91% of the final product. For a 20mer, it is (99%)19 = 81%. This means that, depending on the process used to synthesise a 20mer, a maximum of 81% of the final product will be full length chains and 19% will be truncated sequences. If, for example, lower quality chemicals are used, then the synthesis will be less efficient, perhaps only 98%, resulting in the final result of the 20mer synthesis only being 68% full length product and 32% truncated sequences. Purification makes it possible to separate the full length product from the truncated sequences. Synthesis of longer oligonucleotides and purification For short oligonucleotides, the number of truncated sequences, so called n-1 bands, is relatively low. HPLC purification isn’t necessary for simple applications, because most of the final result of the synthesis is full length product, providing high quality base material is used. If left undone for other applications, however, there might be considerable side effects or even false results, should the truncated sequences trigger a competitive reaction to the lengthened sequences. Quantitative processes are especially sensitive to such influences, which means it is a good idea to conduct either RP-Fast purification or FDC-HPLC. The longer the oligonucleotide becomes in the synthesis, the greater the percentage of n-1 truncated sequences. Taking a 90mer as an example, at 99% synthesis efficiency, the full length product only accounts for 41% of the result. Truncated sequences make up the other 59%. At 98% efficiency, the synthesis theoretically only produces 16.6% full length product. The greater the length, however, the less efficient the synthesis and thus the poorer the yield. Thus, it can be assumed that an unpurified 90mer only contains 10-15% full length product. For a 150mer the share of full length product is less than 1%! If poorer quality chemicals are also used, the share of full length product with the correct sequence decreases even further. Moreover, one mustn’t forget that long oligonucleotide can be very difficult to use because of secondary structures and steric factors. Hence, if long oligonucleotides are to be used in research, it is absolutely necessary to purify them to a great degree and use high quality chemicals. Due to the length, the operating characteristics of the full length product are similar to those of the truncated sequences, which is why the purification programme has to be developed to filter out 90-99.9% of the truncated sequences. Inefficient purification only removes the short failure sequences, leaving long truncated bands in the mix. It is no longer possible to successfully use the oligonucleotide.
Fig. 1 | Purification of standard oligonucleotides. The higher the type of purification is found in the chart, the more effectively n-1 bands are removed from the main product. The purification of the main product also depends on the base length of the oligonucleotide. The longer it is, the more difficult it becomes to filter out the truncated strands during purification. Therefore, longer sequence lengths require more and more complicated purification protocols. The method of purification should be chosen based primarily on the length and application of the oligonucleotide. For a simple PCR reaction, the OE standard is sufficient for a length of 10-45 bases. Depending on the area of application, however, FDC-HPLC might already make sense for a length of 20 bases. _________________________ |
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