Figure 3. Ligation reactions by using N-Cyanoimidazole.

(A) Lane 1: Marker of 5′-P³² labeled P-b; Lane 2: 3′-3′ ligation without N-Cyanoimidazole; Lane 3: 5′-3′ ligation that is similar as in 3′-3′ ligation in length. P-a' has the same sequence with that of P-a in opposite direction; Lane 4: 3′-3′ ligation under standard reaction condition; Lane 5: 5′-3′ ligation which similar as in 5′-5′ ligation in length; Lane 6: 5′-5′ ligation under standard reaction condition; Lane 7: same as lane 6, 5′-5′ ligation by using P-b1 that has one more A than P-b at 5-end. Lane 8: marker of 5′-P³² labeled Template-33. (B) Verification experiments of 3′-3′ chemical ligation. Lane 1: Ligation reaction containing 5′-P32 labeled P-a ligated to 3′-end phosphorylated oligonucleotides P-b in the presence of Template-33; Lane 2: the same reaction as lane 1 but without P-b; Lane 3: the same reaction as lane 1 but without Template-33; Lane 4: Marker of 5′-P³² labeled P-a; Lane 5: Ligation reaction containing 5′-P³² P-b with 3′-phosphorylated oligonucleotides P-a in the presence of Template-33; Lane 6: the same reaction as lane 5 but without P-a; Lane 7: the same reaction as lane 5 but without Template-33; Lane 8: Marker of 5′-P³² labeled P-b; Lane 9: marker. P³² in red indicated the isotope labeling position. The asterisk denotes isotope labeling. Full-length blot images are available in Supplementary Figure S10.