RETRIEVAL OF DNA-SEQUENCES PRESENT AT AN EXTREMELY-LOW-FREQUENCY

We describe a method for retrieving sequences with one or two point mu tations of a given target sequence, which are present in a DNA populat ion at a frequency of 1 in 466 x 10(3) and 1 in 28 x 10(3) molecules, respectively. By stringent hybridization to a stable, chemically immob ilized probe, a large excess of unrelated fragments is removed, and th e bound sequences are dissociated and amplified. By repeating the hybr idization-amplifcation cycles twice, we achieved an estimated enrichme nt of 404 000-fold and 1612-fold, respectively, which was confirmed by cloning the resultant products and sequencing 35 clones. This procedu re can be applied to retrieve mutated sequences that exist at an extre mely low frequency in a DNA population.