IMPROVEMENTS TO THE DIFFERENTIAL DISPLAY METHOD FOR GENE ANALYSIS

Differential display requires two primers to identify discordantly exp ressed mRNAs. Anchored primers with at least one G residue were superi or to those which had one C residue and those ending in A or T were th e least efficient. Arbitrary primers with GC pairs at the 5'end were s uperior to primers with GC pairs at the 3'end. We found that screening of amplified DNA-fixed membranes by a slot blot manifold and hybridiz ation with DNA probes made from sample RNA rapidly re-screened with DN A fragments for differential expression, requires less RNA and is fast er than Northern analysis. Similarly, hybridization of DNA fragments t o plasmid DNA fixed to membranes and direct PCR sequencing rapidly det ermine homology to common laboratory plasmids prior to any further man ipulation. These modifications permit rapid isolation and characteriza tion of DNA fragments identified by differential display. (C) 1994 Aca demic Press, Inc.