Quantifying DNA exonuclease activity using a switchSENSE^® hybridization assay 

The activity of the T5 exonuclease is quantified in a simple and quick workflow. This study demonstrates that the presence of T5 exonuclease down to 0.001 U/µL could be detected and quantified using a switchSENSE^® hybridization assay.
The combined analysis of signal end levels and real-time kinetics enables the user to follow the time course of the enzymatic reaction on the sacrificial DNA and to eventually identify the length of the digested DNA products. This assay is performed in less than 5 minutes and the liquid handling steps required to incubate the enzyme with sacrificial DNA beforehand could be performed automatically by the DRX’s auto-sampler system. The study further expands the possibilities to perform experiments at different temperature and reaction time, which would improve the detection limit. It is also straightforward to adapt the assay to monitor exonucleases with an opposite directionality.