Detection and differential display of expressed genes by DDRT-PCR.

~The first two authors contributed equally to the establishment of the method and to the results obtained. 4present address: Max-Planck-Gesellschaft, MaxDelbr~ick-Haus, 13122 Berlin-Buch, Germany. Analysis of gene expression is a central aim in most studies in molecular and cell biology. Interest in tissue-specific gene expression and, in particular, in changes in the expression patterns occurring in response to either mutations or transfected genes has stimulated the search for proper methods to identify the actual differences between two situations. For a long time, differential hybridization was the only method allowing for isolation of genes that were in either of two situations; there was no method available that would indicate the total number of genes subject to upor down-regulation in a particular setting. Pardee and co-workers were the first to show (1-3) that combinations of arbitrary primers (previously used for amplification of DNA polymorphisms (4's) and for obtaining mRNA tags (6)) with anchored cDNA primers can be applied successfully to generate a set of fragments from cDNA derived from the total mRNA of a cell. Under the appropriate conditions, the pattern of fragments derived from one type of cells is reproducible and can be compared with that of another cell type. (1'3'7) Our theoretical calculations and experimental results confirmed that the method can generate patterns of bands that might represent almost all expressed genes in a particular cell and can also reproducibly detect differences in gene expression between two or more cell types or situations. (7'8) The method was named "differential display ''(~) or DDRT-PCR. (7'8) This method has already been applied successfully by several groups using only a limited set of primers (9-~) and will probably become a standard method for studies on differential gene expression. Here, we present a detailed protocol that is applicable to any set of two or more comparable eukaryotic cell types and will result in reproducible patterns of PCR products that correspond to expressed genes.