A new technique to prevent self-ligation of DNA

The most widely used technique for preventing self-ligation (self-circularization and concatenation) of DNA is dephosphorylation of the 5′-end, which stops DNA ligase from catalyzing the formation of phosphodiester bonds between the 3′-hydroxyl and 5′-phosphate residues at the DNA ends. The 5′-dephosphorylation technique cannot be applied to both DNA species to be ligated and thus, the untreated DNA species remains capable of self-ligation. To prevent this self-ligation, we replaced the 2′-deoxyribose at the 3′-end of the untreated DNA species with a 2′,3′-dideoxyribose. Self-ligation was prevented at the replaced 3′-end, while the 5′-phosphate remaining at the 5′-end permitted ligation with the 3′-hydroxyl end of the 5′-dephosphorylated DNA strand. We successfully applied this 3′-replacement technique to gene cloning, adapter-mediated polymerase chain reaction and messenger RNA fingerprinting. The 3′-replacement technique is simple and not restricted by sequence or conformation of the DNA termini and is thus applicable to a wide variety of methods involving ligation.