Fig. 1. Strategy (Strategic diagram) of preventing adapters self-ligation using methylation-sensitive endonucleases (with CGat adapter as an example).
Three enzymatic sites harboring CGc, CGg and CGt strings are yielded and their compatible ends in the adapters is CGa. Two of CGa with a following T composes a ClaI site when the adapters are self-ligated. However, the ligated products between CGc, CGg and CGt from target nucleic acids and CGa from the adapters form no restriction site and thus the combination of these enzymes ensures a virtually unidirectional reaction of producing ligation between target inserts and their adapters. Here MSRE represents methylation sensitive restriction endonuclease
Fig. 2. Self-ligation of CGat adapters prevented by the addition of endonuclease ClaI.
Fig. 3. Endonuclease ClaI prevented CGat adapters from self-ligation and promoted its ligation with insert
(A) The expected product from the ligation of insert and adapters in lane 5 is obviously more than that observed in lane 4. (B)The ligated products were confirmed by Sanger sequencing.
Fig. 4. Self-ligation of the TAat adapters were inhibited by adding restriction endonuclease AseI.
Fig. 5. Endonuclease AseI and MseI together prevented TAat adapters from self-ligation and promoted its ligation with insert.
(A)The expected band of the insert-adapter ligated product in lane 5 was more than that in lane 4. (B) Sequencing chromatographs of the ligated products from insert and adapters.