Extended Data Fig. 3: One-pot INSPECTR fails due to the incompatibility in reaction rates between splint-ligation and probe double-stranding.
a, Schematic of one-pot ligation-second strand synthesis. If second strand synthesis is faster than the ligase, then the single-stranded junction won’t be accessible for SplintR ligase. Measured both by b, cell-free expression and c, qPCR using primers that span the ligation junction, as well as cell-free expression, the addition of exogenous DNAP, which is necessary to generate a transcribable expression cassette, impedes the splint-ligation of functional probe.
