Substantially enhanced cloning efficiency of SAGE (Serial Analysis of Gene Expression) by adding a heating step to the original protocol

Abstract

The efficiency of the original SAGE (Serial Analysis of Gene Expression) protocol was limited by a small average size of defied concatemers. We describe a modification of the technique that overcomes this problem. Ligation of ditags yields concatemers of various sizes. Small concatemers may aggregate and migrate with large ones during gel electrophoresis. A heating step introduced before gel electrophoresis breaks such contaminating aggregates. This modification yields cloned concatemers with an average size of 67 tags as compared to 22 tags by the original protocol. It enhances the length of cloned concatemers substantially and reduces the costs of SAGE.