A new universal linker for solid phase DNA synthesis.

Abstract

A method is described as an alternative to the use of nucleoside pre-functionalized supports for DNA synthesis. The procedure should allow the generation of 3'-OH terminal moieties of any natural or modified DNA fragment using a single derivatized solid support material. The method utilizes 1-O-(4,4' dimethoxytrityl)-2- O-succinoyl-3-N-allyloxycarbonylpropane immobilized on amino-propyl CPG followed by subsequent coupling of unit phosphoramidites. Work up is accomplished by removal of the 3-N-allyloxycarbonyl group [Pd(0) at 50 degrees C for 15 min] followed by cleavage under very mild conditions (aqueous TEAA/NH3 buffer pH 10, room temperature) to release the desired product. The mechanism is believed to involve nucleophilic attack of the linker-derived amino group on the 3'-phosphate triester, followed by elimination of the desired product. DNA synthesis with the new support and with classical nucleotide synthesis supports have been performed, and the products shown to be identical. Further proof of product integrity was given by MALDI mass spectral studies and the efficacy of DNA primers made with the new support in PCR amplification.