Figure 5
From: Modular affinity-labeling of the cytosine demethylation base elements in DNA
(a) Steps of the labeling scheme. (i)–(iii) the same as in Figs. 2 and 3. “Lig” indicates nick ligation. (b–c) Denaturing gel analyses of 34 nt DNA constructs featuring a single 5caC at base position 22. In each, the base is excised with WT TDG and the construct is treated with EndoIV to prepare the 3′ end of the gap, EndoVIII to remove the phosphate flap, T4 polymerase and biotinylated dCTP to label, and T4 ligase to repair the remaining nick. In (b), tightly-bound WT TDG is removed using APE1 D308A and in (c) it is removed by phenol exposure. Lane (i): annealed oligonucleotide; lane (ii): following glycosylase, APE1/phenol treatment, and endonuclease; lane (iii): following polymerase fill-in with a biotinylated nucleotide; lane labeled “Lig” is post ligation yielding a biotin-labeled construct with a repaired backbone (red). Construct lengths at left apply to both gels and * indicates DNA length plus biotin tag. Directly below lanes (ii), (iii), and “Lig” are listed target product yields from the previous step followed by the net yield in red. Full gels are shown in Supplementary Figure S6.
