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 characterization of genes with specific spatial and temporal expression patterns. Several methods have been developed to identify such DNA sequences. The most comprehensive technique involves the genomewide probing of DNA sequence microarrays with mRNA sequences. However, at present this technology is limited 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 differentially expressed mRNA species. The method involves the competitive hybridization of an excess of driver cDNA fragments (D) to a trace of tester cDNA fragments (T), and the subsequent purification of tester homohybrids (in which both strands are contributed by the tester cDNA). After competitive hybridization, 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 requires highly efficient procedures. In SABRE, the isolation of tester homohybrids is afforded by a combination of three successive steps: removal of biotinylated terminal sequences from most of the heterohybrids by S1 nuclease digestion, capture of biotinylated hybrids with streptavidin-coated paramagnetic beads, and specific release of homohybrids from the beads by restriction nuclease digestion. If several rounds of SABRE selection are performed in series, even relatively rare differentially expressed mRNA sequences may result in the production of predominant cDNA fragments in the final tester homohybrid population.
Schibler
U
U
Department of Molecular Biology, University of Geneva, 30, Quai Ernest Ansermet, CH-1211 Geneva-4, Switzerland. ueli.schibler@molbio.unige.ch
Rifat
D
D
Lavery
D J
DJ
D000818:Animals; D001710:Biotin / Q000378:metabolism*; D019921:Biotinylation; D004262:DNA Restriction Enzymes / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D020869:Gene Expression Profiling / Q000379:methods*; D007700:Kinetics; D051379:Mice; D008957:Models, Genetic; D009693:Nucleic Acid Hybridization / Q000379:methods; D020411:Oligonucleotide Array Sequence Analysis; D012333:RNA, Messenger / Q000378:metabolism*
D018076:DNA, Complementary; D012333:RNA, Messenger; D001710:Biotin; D004262:DNA Restriction Enzymes