A NEW UNIVERSAL LINKER FOR SOLID-PHASE DNA-SYNTHESIS

A method is described as an alternative to the use of nucleoside pre-f unctionalized supports for DNA synthesis, The procedure should allow t he generation of 3'-OH terminal moieties of any natural or modified DN A fragment using a single derivatized solid support material, The meth od utilizes 1-O-(4,4'dimethoxytrityl)-2-O-succinoyl- 3-N-allyloxycarbo nylpropane immobilized on amino-propyl CPG followed by subsequent coup ling of unit phosphoramidites, Work up is accomplished by removal of t he 3-N-allyloxycarbonyl group [Pd(0) at 50 degrees C for 15 min] follo wed by cleavage under very mild conditions (aqueous TEAA/NH3 buffer pH 10, room temperature) to release the desired product. The mechanism i s believed to involve nucleophilic attack of the linker-derived amino group on the 3'-phosphate triester, followed by elimination of the des ired product, DNA synthesis with the new support and with classical nu cleotide synthesis supports have been performed, and the products show n to be identical. Further proof of product integrity was given by MAL DI mass spectral studies and the efficacy of DNA primers made with the new support in PCR amplification.