Fig. 4.

Ice eutectic phase activation enables a complete pathway from unactivated mononucleotides through activation to primer extension. (A) Schematic of primer-template complex. (B) Time course of primer extension using pure activated mononucleotides (5 mM *pG and 5 mM *pC, room temperature). (C) Time course of primer extension reactions using pNs (n = C and G) generated with MeNC activation chemistry under ice eutectic phase. (D) Primer-template was added to already-activated mononucleotides generated by MeNC-based nucleotide activation under ice eutectic phase conditions, yielding extended primer (as in C). (E) Primer-template duplex was directly treated with pNs (n = C and G) and MeNC activation chemistry under ice eutectic phase. The products of the reaction were then incubated for an additional 24 h at room temperature (F) without and (G) with bridge-forming activation. Each set of three lanes in (D–G) represents independent experimental replicates under the indicated condition. (H) Quantification of primer extension products. Positions of primer and +1 to +3-nucleotide extension products are indicated. Gel image of replicates for (B, C) is included in SI Appendix, Fig. S7.