Improved NlaIII digestion of PAGE-purified 102 bp ditags by addition of a single purification step in both the SAGE and microSAGE protocols

摘要

Despite the success of microarray technologies, serial analysis of gene expression (SAGE) still remains the only technique that allows an accurate quantitative and qualitative analysis of cell transcription in a variety of physiological and pathological conditions. Nevertheless, the efficiency of SAGE is limited by the numerous gel purification steps required and these increase the possibility of contamination and reduce or inhibit the activity of the enzymes used in the protocol. In order to eliminate this problem, we have modified the original protocol by adding a single purification step before NlaIII digestion of the ditags. This allows us to increase the yield of digested ditags without reducing the amount of DNA or affecting the subsequent concatemerization.