IDENTIFYING DIFFERENCES IN MESSENGER-RNA EXPRESSION BY REPRESENTATIONAL DIFFERENCE ANALYSIS OF CDNA

Detection of differentially regulated genes has been severely hampered by technical limitations. In an effort to overcome these problems, th e PCR-coupled subtractive process of representational difference analy sis (RDA) [Lisitsyn,N. at al. (1993) Science 259, 946 - 951] has been adapted for use with cDNA, In a model system, RAG-1 and RAG-2 the gene s responsible for activating V(D)J recombination, were identified in a genomic transfectant by cDNA RDA in a small fraction of the time take n by conventional means, The system was also modified to eliminate exp ected difference products to facilitate the identification of novel ge nes. Additional alterations to the conditions allowed isolation of dif ferentially expressed fragments. Several caffeine up-regulated clones were obtained from the pre-B cell line 1-8, including IGF-1B, and a pr edicted homologue of the natural killer cell antigen, NKR-P1. The appr oach was found to be fast, extremely sensitive, reproducible, and pred ominantly lacked false positives, cDNA RDA has the capacity and adapta bility to be applied to a wide range of biological problems, including the study of single gene disorders, characterization of mutant and co mplemented cell types, developmental or post-event expression time cou rses, and examination of pathogen - host interactions.