Correlation between lesion size, biotin incorporation, and plasmid recovery.

<p>(<b>A</b>) Incorporation of 1 or 2 biotinylated dCTP molecules is sufficient to recover the targeted plasmid by streptavidin purification. Schematic of the experiment is depicted at the top of the figure. Plasmids were untreated (scDNA) or nicked at one position with Nt.BsmAI (nicked DNA), followed by treatment with Klenow for 30 min at 25°C in the presence or absence of dTTP (dT) and varying ratios of biotinylated (bio-dC) and normal dCTP (dC). As indicated in the schematic, the lack of dGTP and dATP only allowed for the incorporation of 1 or 2 bio-dC molecules per plasmid. The bars (fold change) represent the difference between the qPCR C_t value for each sample before and after the streptavidin purification, normalised to the sample (scDNA or nicked DNA) treated with the Klenow and bio-dC (no dT and set to 1). Error bars indicate ± SD (n = 3). Time line of the experiment is shown above the graph. (<b>B</b>) Patch length of incorporated bio-dC does not bias the recovery of plasmids from IVR. The amount of bio-dC incorporation depending on the presence of other dNTPs was monitored by an AID-induced IVR assay. The fold change represents the difference between the qPCR C_t value of each sample normalised to the FE-treated sample (no G-AID) that was set to 1.