Figure 4.

Optimization of the ring closure ligation. A modified pET11aΔH intermediate (Fig. 1, structure 4) was incubated with T4 DNA ligase using DNA concentrations decreasing from 100 to 10 ng/µl (see Materials and Methods for conditions). This LS has one DNA end generated by EcoRI digestion and one contributed by the T·G-10H oligonucleotide heteroduplex. The supercoiled (SC) pET11aΔH plasmid loaded in the first and last lanes contains a small amount of NC molecules that serves as a close marker for the mobility of the desired product. At 100 ng/µl DNA, the predominant products are multimers that migrate more slowly. At 10 ng/µl template, the desired NC heteroduplex represents up to 30% of the products, and the formation of linear multimers is reduced. For each lane representing a sample of a ligation reaction, an equivalent sample was treated with Exonuclease V, which degrades linear molecules and reveals the circular products. Note that a trace amount of CD is formed in this experiment.