Figure 3. Template tethering.

(A) Illustration of competimer oligonucleotide function. Unligated RCOs are tethered to an unligated BCO via the template DNA during bead hybridization (top). A competimer oligonucleotide displaces the RCO from the bead-bound species. (B–E) Demonstration of template tethering and effects of a competimer oligonucleotide (all +/−s.d., N = 4 independent experiments). Template DNA (2 ng/µl) was diluted with λ-phage DNA at the same concentration. Ligase discrimination reactions were assembled and subjected to 300 thermal cycles or no thermal cycling (mock). Grey fill indicates the 95% CI calculated from the incorrect signal. (B) Mock series demonstrates false signal in the absence of ligase activity. (C) Reactions subjected to thermal cycling demonstrates SNP discrimination (blue) and incorrect signal similar to panel B (orange). (D) Mock series subjected to bead hybridization with 25 nM competimer demonstrates false signal suppression. (E) Reactions subjected to thermal cycling and bead hybridization with 25 nM competimer lack the false signal (orange) shown in panel C. Data for 0.1 ng template input were not significantly different than the same data point in panel C (open blue square in both panels; MWW p = 0.47).