Rapid isolation of cosmid insert DNA by triple-helix-mediated affinity capture.

A simple and rapid method for the isolation of cosmid insert DNA was developed based on triple-helix-mediated affinity capture (TAC). A modified cosmid was constructed from the SuperCos 1 cosmid vector by flanking the cloning site with two homopurine-homopyrimidine triple-helix-forming sequences. The cosmid DNA is digested with NotI restriction enzyme to release the insert DNA. The NotI-digested cosmid DNA is then combined with a biotinylated homopyrimidine oligonucleotide in an acidic buffer solution to form a triple-helix complex. The triple-helix complex is captured with streptavidin-coated magnetic beads. Insert DNA is eluted by adding a pH 9 buffered solution to the captured complex. The purified insert DNA is recovered with a yield of up to 95% and a purity of at least 95%. The isolated insert DNA was directly digested with CviJI restriction endonuclease to generate random fragments for shotgun sequencing.