GENERATION OF MULTIPLE MESSENGER-RNA FINGERPRINTS USING FLUORESCENCE-BASED DIFFERENTIAL DISPLAY AND AN AUTOMATED DNA SEQUENCER

Differential display is a method for the survey, analysis and comparis on of gene expression in eukaryotic cells and tissues. Differential di splay involves isolation of high-quality nondegraded RNA, selective re verse transcription of plyadenylated mRNA using specific anchored olig opoly deoxythymidine [oligo(dT)] primers, and the subsequent PCR ampli fication of the cDNA with the same oligo(dt), an arbitrary upstream pr imer and radioisotopes for labeling the PCR products. The radioisotopi cally labeled products are then separated on a sequencing gel. In this report, we describe a rapid specific, nonradioactive fluorescent diff erential display methodology in which fluorescently differentially lab eled anchored oligo(dT) downstream primers are used in the reaction, w ith subsequent analysis of fluorescently labeled PCR products on ail a utomated sequencer: Complete gene expression profiles, containing mult iple mRNA fingerprints are possible by the simultaneous comparison of the multicolored banding patterns of the fluorescently differentially labeled products from several primer combinations. This modification o f the differential display technique simplifies the assay and increase s the throughput of high sample volumes required for comparative gene expression studies in various clinical applications.