The isolation of differentially expressed mRNA sequences by selective amplification via biotin and restriction-mediated enrichment

Molecular analysis of development frequently implies the isolation and char acterization of genes with specific spatial and temporal expression pattern s. Several methods have been developed to identify such DNA sequences. The most comprehensive technique involves the genomewide probing of DNA sequenc e microarrays with mRNA sequences. However, at present this technology is l imited to the few organisms for which the entire genome has been sequenced. Here, we describe a subtractive hybridization technique, called selective amplification via biotin and restriction-mediated enrichment (SABRE), which allows the selective amplification of cDNA fragments representing differen tially expressed mRNA species, The method involves the competitive hybridiz ation of an excess of driver cDNA fragments (D) to a trace of tester cDNA f ragments (T), and the subsequent purification of tester homohybrids (in whi ch both strands are contributed by the tester cDNA). After competitive hybr idization, cDNA fragments that are more abundant in the tester than in the driver are enriched in the tester homohybrids. However, as the fraction of tester homohybrids is very small [T-2/(D + T)(2)], their purification requi res highly efficient procedures, In SABRE, the isolation of tester homohybr ids is afforded by a combination of three successive steps: removal of biot inylated terminal sequences from most of the heterohybrids by S1 nuclease d igestion, capture of biotinylated hybrids with streptavidin-coated paramagn etic beads, and specific release of homohybrids from the beads by restricti on nuclease digestion. If several rounds of SABRE selection are performed i n series, even relatively rare differentially expressed mRNA sequences may result in the production of predominant cDNA fragments in the final tester homohybrid population. (R) 2001 Academic Press.