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Library screening methods based on nucleotide sequences usually employ in situ filter hybridization to colonies and plaques (1). These methods arc useful and reliable, but involve labor-intensive steps like filter preparation and manipulation and the re-isolation of positive clones from the original bacterial plates. Triplex affinity capture (TAC) is an alternative screening method based on direct DNA isolation (2). TAC was used to isolate microsatellite clones from a human genomic library as potentially useful DNA markers for genetic linkage mapping (2). In TAC, plasmid DNA prepared en masse from a library is incubated with a biotinylated homopyrimidine oligonucleotide probe under mildly acidic conditions to form an intermolecular triplex between the target DNA and the probe. Subsequently, streptavidin-coated magnetic beads are used to capture this complex and separate it from bulk DNA. Double-stranded target DNA is recovered by incubating the beads under mildly alkaline conditions that selectively destabilize the triple helix but not the Watson -Crick double helix. Since TAC is performed in several small tubes, large numbers of clones or several libraries may be screened simultaneously. TAC yields intact, double-stranded DNA that can readily be used for enzyme digestion, electrophoresis or transformation.

Triplex affinity capture of a single copy clone from a yeast genomic library