Figure 4.

HPR forms mainly or exclusively 3′–5′ linkage upon ligation under freezing conditions. Lane 1, ligation substrates 3′-LS and 5′-LS obtained by HPR cleavage under standard solution conditions (37°C/10 mM Mg²⁺) and gel-purified. Lane 2, ligation of the 3′-LS and 5′-LS by the HPR under freezing conditions (–10°C), 14 h. Lane 3, gel-purified ligation product LP. Lane 4, cleavage of the LP by the HPR under standard solution conditions (37°C/10 mM Mg²⁺), 2 h.