Gilar M
Waters Corporation, Milford, MA 01757, USA.
J Chromatogr A. 2000 Aug 18;890(1):167-77. doi: 10.1016/s0021-9673(00)00521-5.
Purification of target oligodeoxyribonucleotides from failure sequence by-products of synthesis is often required for polymerase chain reaction primers, DNA sequencing and other oligonucleotide applications. We have developed purification protocols based on a reversed-phase mechanism ("trityl on" purification) using a 96-well Oasis HLB extraction plate. The Oasis HLB sorbent combines excellent pH stability with a high loading capacity allowing for single-step purification of 0.2 microM scale synthesis. After sample loading and washing, the oligonucleotide trityl group is cleaved on the plate with 2% trifluoroacetic acid. Target DNA is eluted with acetonitrile-0.36 mM triethylamine acetate, pH 11.3 (10:90, v/v). Typical yield of purified product is 60-95%. Final purity, measured by capillary gel electrophoresis, was found to be 90% or greater. Alternatively, highly pure oligonucleotides can be obtained by a RP-HPLC "trityl off" method using an XTerra C18 column. The use of volatile triethylamine acetate buffer as an ion-pair for RP-HPLC eliminates the need for further desalting.
对于聚合酶链反应引物、DNA测序及其他寡核苷酸应用而言，常常需要从合成失败序列副产物中纯化目标寡脱氧核糖核苷酸。我们基于反相机制（“三苯甲基在位”纯化）开发了纯化方案，使用96孔Oasis HLB萃取板。Oasis HLB吸附剂兼具出色的pH稳定性和高负载量，可实现0.2微摩尔规模合成的单步纯化。样品加载和洗涤后，用2%三氟乙酸在板上裂解寡核苷酸三苯甲基基团。目标DNA用乙腈-0.36 mM乙酸三乙胺（pH 11.3，10:90，v/v）洗脱。纯化产物的典型产率为60%-95%。通过毛细管凝胶电泳测定，最终纯度为90%或更高。或者，可使用XTerra C18柱通过RP-HPLC“三苯甲基脱除”方法获得高纯度寡核苷酸。使用挥发性乙酸三乙胺缓冲液作为RP-HPLC的离子对，无需进一步脱盐。
