Retrieval of DNA Sequences Present at an Extremely Low Frequency

Abstract

We describe a method for retrieving sequences with one or two point mutations of a given target sequence, which are present in a DNA population at a frequency of 1 in 466 × 103 and 1 in 28 × 103 molecules, respectively. By stringent hybridization to a stable, chemically immobilized probe, a large excess of unrelated fragments is removed, and the bound sequences are dissociated and amplified. By repeating the hybridization-amplification cycles twice, we achieved an estimated enrichment of 404000-fold and 1612-fold, respectively, which was confirmed by cloning the resultant products and sequencing 35 clones. This procedure can be applied to retrieve mutated sequences that exist at an extremely low frequency in a DNA population.