Improvements to the differential display method for gene analysis.

Abstract

Differential display requires two primers to identify discordantly expressed mRNAs. Anchored primers with at least one G residue were superior to those which had one C residue and those ending in A or T were the least efficient. Arbitrary primers with GC pairs at the 5'end were superior to primers with GC pairs at the 3'end. We found that screening of amplified DNA-fixed membranes by a slot blot manifold and hybridization with DNA probes made from sample RNA rapidly re-screened with DNA fragments for differential expression, requires less RNA and is faster than Northern analysis. Similarly, hybridization of DNA fragments to plasmid DNA fixed to membranes and direct PCR sequencing rapidly determine homology to common laboratory plasmids prior to any further manipulation. These modifications permit rapid isolation and characterization of DNA fragments identified by differential display.