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.