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.
Ukai
Hideki
H
Japan Science and Technology Corporation, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba, 263-8555, Japan.
Ukai-Tadenuma
Maki
M
Ogiu
Toshiaki
T
Tsuji
Hideo
H
D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D004249:DNA Damage / Q000187:drug effects / Q000235:genetics; D016172:DNA Fingerprinting / Q000379:methods*; D011088:DNA Ligases / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D005786:Gene Expression Regulation; D008956:Models, Chemical; D008957:Models, Genetic; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis
D012333:RNA, Messenger; D004247:DNA; D011088:DNA Ligases