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.
Ji
H
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Department of Chemistry, University of Wisconsin-Madison 53706.
Smith
L M
LM
Guilfoyle
R A
RA
D001483:Base Sequence; D003001:Cloning, Molecular; D003360:Cosmids / Q000235:genetics* / Q000302:isolation & purification*; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification*; D015723:Gene Library; D005821:Genetic Techniques; D005822:Genetic Vectors; D015894:Genome, Human; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction
D004247:DNA