Retrieval of DNA sequences present at an extremely low frequency.
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.
Zafiropoulos
A
A
Institute of Molecular Biology and Biotechnology, Heraklion, Crete, Greece.
Hatzidakis
G
G
Mavrogiannis
L
L
Klinakis
A
A
Kandilogiannaki
M
M
Krambovitis
E
E
D003001:Cloning, Molecular; D004247:DNA / Q000302:isolation & purification*; D005787:Gene Frequency; D015723:Gene Library; D005813:Genes, Synthetic / Q000276:immunology; D015699:HIV Envelope Protein gp120 / Q000235:genetics; D015497:HIV-1 / Q000235:genetics; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics; D009693:Nucleic Acid Hybridization; D010446:Peptide Fragments / Q000235:genetics; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D017422:Sequence Analysis, DNA / Q000379:methods*
D015699:HIV Envelope Protein gp120; C059762:HIV envelope protein gp120 (305-321); D007135:Immunoglobulin Variable Region; D010446:Peptide Fragments; D004247:DNA