FIG. 5.

Ligation activity of LdMNPV ligase on three synthetic double-stranded DNA substrates. (a) The end-labeled (indicated by the asterisk) donor 18-mer and the three acceptor 18-mers are shown relative to the 36-mer complementary strand (adapted from the work of Ho et al. [17]). Duplex substrates were prepared as described in Materials and Methods. (b) A standard reaction mixture (20 μl) containing unlabeled ATP and 0.9 pmol of ³²P-labeled DNA substrate, containing either a nick, a 1-nt gap, or a 2-nt gap, with or without 50 ng of ligase was incubated at 20°C for 30 min and subjected to electrophoresis on a 12.5% polyacrylamide–7 M urea gel. The results shown are as follows: lane 1, ³²P-5′-end-labeled 18-mer donor strand; lanes 2 and 3, nicked substrate without (lane 2) or with (lane 3) ligase; lanes 4 and 5, 1-nt-gap substrate without (lane 4) or with (lane 5) ligase; lanes 6 and 7, 2-nt-gap substrate without (lane 6) or with (lane 7) ligase. The locations of the 10-bp-ladder (Gibco BRL) size standards are indicated on the left in base pairs.