Scheme 2. Schematic representation of the HPA for S. aureus DNA ligase.

After ligation of the AE-labelled DNA substrate, the AE label is rendered less susceptible to alkaline hydrolysis. The label from non-ligated DNA is preferentially hydrolysed by mild alkaline stop/selection reagent [360 mM Na₂B₄O₇ containing 240 mM HCl and 0.07% (v/v) Triton X-102, pH 8] with incubation at 40 °C in a water bath or heating block. Addition of 0.5 M NaOH containing 0.3% (v/v) H₂O₂ results in the decomposition of the AE label from ligated DNA and release of light at 430 nm (chemiluminescence).