A Direct-Sequencing-Based Strategy for Identifying and Cloning cDNAs from Differential Display Gels

This report describes an approach to identifying and cloning messages from differential display (DD) gels that has markedly reduced the occurrence of false positives as well as the time required for the process. Most importantly, the use of Northern blots with potentially contaminated probes is avoided and a streamlined direct sequencing protocol is used as an initial step. The individual methods presented here are not new, but have been put together from a variety of sources in a revised order. Methods for the DD reverse transcription-polymerase chain reactions (RT-PCR) and electrophoresis conditions, for which we have used published methods (1 ) and the GenomyxLR DNA sequencer (see Note 1 and ref. 2 ), are not covered here.