Analysis of differential display RT-PCR products using fluorescent primers and GENESCAN software

Differential display reverse transcription PCR (DDRT-PCR) is a procedure used to identify the induction or repression of gene expression. In most DDRT-PCR protocols, radioisotopes are incorporated during PCR and the cDNA products are detected by autoradiography. This report describes the fluorescent labeling of cDNAs and their detection on automated sequencers from PE Applied Biosystems. A fluorescent tag can be incorporated into the PCR product by using either a labeled primer or a labeled dUTP. The fluorescent signals are analyzed by GENESCAN software. Fluorescent DDRT-PCR increases throughput and obviates the handling of hazardous radioisotopes. A PCR cycling profile, expected to give improved reproducibility, is also described. Because amplified cDNAs can't be recovered from the automated sequencer gel, suggestions are given for the identification and recovery of differentially expressed cDNAs.