Fig. 3

Proof-of-principle experiments. a Generation of Full-records requires the colocalization of probes, here by biotinylated hairpin loops bound to streptavidin, whereas isolated probes can always generate Half-records. Cropped denaturing PAGE gel depicting 10 μl reactions (40 min at 37 °C) with biotin–streptavidin association and 4:1 overall probe/streptavidin stoichiometry (inset), 8 and 22 nt barcodes (19 and 33 nt stem lengths copied), 10:1 initial primer/probe, and 40 nM total probe concentration. A single primer sequence was used and no secondary amplification was performed. b Auto-cycling is demonstrated by quantification of Cy5-labeled probes on cropped denaturing PAGE gels. Rapidly cycling probes with Iso-dC/dG stoppers and phosphorothioate primers were used, with probes at 0.1 nM and primers at 1000-fold excess to probes in each time series. Quantification of Full-records yields the plot. Half-records are difficult to detect because of low probe concentration. See probe details and sequences for a, b in Supplementary Fig. 2 and full gels in Supplementary Fig. 3. c An example of a single probe (with Barcode i) making multiple partnerships (with Barcodes \({j^*}\)), read with Illumina MiSeq next-generation sequencing. Here, probes encoded a universal primer sequence and unique barcodes (in place of spacer s of Fig. 2a), and were held in tetramers by streptavidin. Primer sequences cropped for clarity. See Supplementary Fig. 4 for the unique-barcode APR cycle, probe details, and sequencing methods