Rapid purification of double-stranded DNA by triple-helix-mediated affinity capture
- Univ. of Wisconsin, Madison (United States)
A simple and rapid method for the preparation of highly pure plasmid DNA has been developed. The DNA is directly captured from bacterial cell lysates by formation of a triple-helical structure between the plasmid dsDNA and a 20-base biotinylated oligonucleotide attached to streptavidin-coated magnetic beads and then eluted from the beads in pH 9 buffer solution. No phenol extraction, ethanol precipitation, RNase digestion, or CsCl gradient centrifugation is required. A general purpose cloning vector, pHJ19, was constructed for this application from pUC19 DNA by insertion of a 40-base sequence suitable for triple-helix formation. The approach was also found suitable for the purification of [lambda] bacteriophage DNA. 32 refs., 6 figs., 1 tab.
- DOE Contract Number:
- FG02-90ER61026
- OSTI ID:
- 6012465
- Journal Information:
- Analytical Chemistry (Washington); (United States), Vol. 65:10; ISSN 0003-2700
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
37 INORGANIC
ORGANIC
PHYSICAL AND ANALYTICAL CHEMISTRY
DNA
PURIFICATION
AFFINITY
BACTERIA
BACTERIOPHAGES
CHROMOSOMES
DNA-CLONING
MOLECULAR STRUCTURE
CLONING
DNA HYBRIDIZATION
HYBRIDIZATION
MICROORGANISMS
NUCLEIC ACIDS
ORGANIC COMPOUNDS
PARASITES
VIRUSES
550200* - Biochemistry
400100 - Analytical & Separations Chemistry