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Retrieval of DNA sequences present at an extremely low frequency.
- Source :
-
BioTechniques [Biotechniques] 1997 Dec; Vol. 23 (6), pp. 1104-9. - Publication Year :
- 1997
-
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.
- Subjects :
- Cloning, Molecular
Gene Library
Genes, Synthetic immunology
HIV Envelope Protein gp120 genetics
HIV-1 genetics
Humans
Immunoglobulin Variable Region genetics
Nucleic Acid Hybridization
Peptide Fragments genetics
Point Mutation
Polymerase Chain Reaction methods
DNA isolation & purification
Gene Frequency
Sequence Analysis, DNA methods
Subjects
Details
- Language :
- English
- ISSN :
- 0736-6205
- Volume :
- 23
- Issue :
- 6
- Database :
- MEDLINE
- Journal :
- BioTechniques
- Publication Type :
- Academic Journal
- Accession number :
- 9421644
- Full Text :
- https://doi.org/10.2144/97236rr02