A new method, affinity capture electrophoresis (ACE), has been developed for the sequence-specific isolation of DNA. The target DNA is complexed with a biotinylated probe and electrophoresed in a gel equipped with a trap of immobilized streptavidin. This selectively captures the target molecule and its biotinylated probe, while other nontarget molecules pass through the trap. The target DNA is subsequently recovered from the trap by destroying the interaction between the target DNA and the biotinylated probe. Two variations of this technique, one using triple-helix formation and the other using hybridization with a uracil-containing DNA probe at the end of the target fragment, proved effective in model experiments. Since this technique requires no denaturation and handles DNA inside an agarose gel matrix, it is, in principle, applicable to the isolation of very large DNAs.