Useful properties of restriction enzymes that recognize interrupted palindromes.

ed here improves upon the concept of directional cloning without forfeiting any of its advantages. The advantages include single-step cloning, high-efficiency generation of the desired recombinant molecule and low background self-ligation of vector. The added benefits of the triple-ligation strategy include easy resolution of vector fragments for gel purification, the ability to invert an insert relative to the orientation of polylinker sites, the ability to flank an insert with the same restriction site on both sides and the ability to use overlapping or otherwise refractory polylinker sites. Note added in proof: The authors wish to acknowledge the similar treatment of the triple-ligation strategy published recently by Zeng et al. (4).