Rapid automated synthesis via diisopropyl phosphoramidite in situ activation. Chemical synthesis and cloning of a calmodulin gene.

Abstract

A gene coding for a calmodulin was synthesized and cloned. The chemical synthesis of the gene, coding for 149 amino acids, was achieved by the enzymatic ligation of 61 chemically synthesized DNA fragments. The DNA fragments were synthesized using a solid support with a diisopropyl phosphoramidite intermediate and in situ activation. The automated standard cycle time was 10 min/addition. The synthesizer was designed and constructed from inexpensive, readily available parts and controlled by a Commodore 64 computer. The gene possesses 27 unique, regularly spaced, restriction endonuclease cleavage sites to facilitate gene mutagenesis.