"In-gel" purified ditags direct synthesis of highly efficient SAGE Libraries.

Abstract

BackgroundSAGE (serial analysis of gene expression) is a recently developed technique for systematic analysis of eukaryotic transcriptomes. The most critical step in the SAGE method is large scale amplification of ditags which are then are concatemerized for the construction of representative SAGE libraries. Here, we report a protocol for purifying these ditags via an 'in situ' PAGE purification method. This generates ditags free of linker contaminations, making library construction simpler and more efficient.ResultsDitags used to generate SAGE libraries were demarcated 'in situ' on preparative polyacrylamide gels using XC and BPB dyes, which precisely straddle the ditag band when a 16% PAGE gel (19:1 acrylamide:bis, 5% cross linker) is used to resolve the DNA bands. Here, the ditag DNA was directly excised from gel without visualization via EtBr or fluorescent dye staining, resulting in highly purified ditag DNA free of contaminating linkers. These ditags could be rapidly self ligated even at 4°C to generate concatemers in a controlled manner, which in turn enabled us to generate highly efficient SAGE libraries. This reduced the labor and time necessary, as well as the cost.ConclusionsThis approach greatly simplified the ditag purification procedure for constructing SAGE libraries. Since the traditional post-run staining with EtBr or fluorescent dyes routinely results in cross contamination of a DNA band of interest by other DNA in the gel, the dry gel DNA excision method described here may also be amenable to other molecular biology techniques in which DNA purity is critically important.