Retrieval of DNA sequences present at an extremely low frequency.

Retrieval of DNA sequences present at an extremely low frequency.

Biotechniques. 1997 Dec;23(6):1104-9. Unique Identifier : AIDSLINE MED/98083381
Zafiropoulos A; Hatzidakis G; Mavrogiannis L; Klinakis A; Kandilogiannaki M; Krambovitis E; Institute of Molecular Biology and Biotechnology, Heraklion, Crete,; Greece.

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 x 10(3) and 1 in 28 x 10(3) 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 404,000-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.

Keywords: *DNA/ISOLATION & PURIF *Gene Frequency *Sequence Analysis, DNA/METHODS
980430
M9841858

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