Analysis of Differential Display RT-PCR Products Using Fluorescent Primers and GENESCAN~(TM)Software

摘要

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 autoradio-graphy. This report describes the fluorescent labeling of cDNAs and their detection on automated sequencers from PE Applied Biosys-tems. 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~(TM)software. Fluorescent DDRT-PCR increases throughput and obviates the handling of hazardous radioisotopes, A PCR cycling profile, expected to give improved re-producibility, 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.