A simple and efficient method for the isolation of differentially expressed genes

A simple and reproducible general approach for the isolation of differentia lly expressed genes is described. Digestion of cDNAs with a class IIs restr iction endonuclease produces fragments with every combination of possible b ases in the cohesive ends. Under stringent conditions, the specific ligatio n of adaptors with perfectly complementary overhangs partitions the cDNA fr agments into non-overlapping subpopulations. Internal cDNA restriction frag ments are exponentially amplified by adaptor primer PCR and visualised by n on-denaturing polyacrylamide gel electrophoresis. The power of the technolo gy was demonstrated using a rat model of pressure-induced left-ventricular hypertrophy (LVH). A set of 29 fragments, derived from a sample (6%) of the possible adaptor pool combinations, displayed apparent differential expres sion. The differential expression of 19 (66%) were confirmed by Northern bl ot analysis. Sequence analysis identified both genes known to be upregulate d in LVH, and novel genes. The fidelity of adaptor ligation was demonstrate d by the isolation of known gene fragments by appropriate adaptor combinati ons. The spiking of mRNA populations with known amounts of a synthetic mRNA demonstrated a current sensitivity equivalent to the detection of transcri pts expressed at the level of as little as 1 in 10,000 molecules. (C) 1998 Academic Press.