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. 2019 Mar 14;47(11):e61. doi: 10.1093/nar/gkz171

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© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 4. — Assay for DNA ligase activity. Incubation of an immobilized hairpin loop oligonucleotide substrate containing a single ligatable nick in one strand within the double-stranded region with T4 DNA ligase, in the presence of ATP, led to an increase in intact substrate retained in the microplate wells following neutral denaturation that was dependent on the concentration of the polymerase (A). Incubation with HepG2 nuclear extract (B) and peripheral blood mononuclear cell pooled nuclear extract (C) also caused concentration-dependent increases in the amount of intact substrate retained in the wells. The apparent DNA ligase activity was directly proportional to the concentration of HepG2 (D) and PBMC (E) nuclear extract over the range 0–0.5 μg/well for the HepG2 nuclear extract (r² = 0.99) and over the range 0–0.63 μg/well for the peripheral blood mononuclear cell nuclear extract (r² = 0.99). Data shown represent the mean ± SD of triplicate technical replicates.