Assembly of linear functional expression elements with DNA fragments digested with asymmetric restriction endonucleases

Abstract

A three-step method is described to assemble linear functional expression elements for rapid gene expression. In step one, a eukaryotic promoter, an open reading frame and a transcription terminator are PCR-amplified individually with primers containing recognition sites of asymmetric restriction endonucleases. In step two, the three DNA fragments, digested with corresponding restriction endonucleases, are directionally joined by T4 DNA ligase to assemble linear functional expression elements. In step three, the resultant linear functional expression elements are amplified with element-specific primers by a second round PCR, followed by transfection into Chinese hamster ovary cells for gene expression.