Rapid purification of double-stranded DNA by triple-helix-mediated affinity capture.
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.
Department of Chemistry, University of Wisconsin-Madison 53706.
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D001426:Bacterial Proteins / Q000737:chemistry; D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000302:isolation & purification*; D004279:DNA, Viral / Q000737:chemistry / Q000302:isolation & purification; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D019809:Streptavidin
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004279:DNA, Viral; D009838:Oligodeoxyribonucleotides; D019809:Streptavidin