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Hijacking a real time detection thermocycler for enzymology: Improvement of a fluorescent bulk assay monitoring helicase activity.
Biochimie.
Helicases are enzymes involved in all aspects of nucleic acid synthesis,regulation and degradation. As a consequence, several methods were developed tomonitor their enzymatic activity. In this report, we described an improvement ofbulk fluorescent helicase assays to overcome their specific limitations (cost,health and safety regulations, etc.). Using a real time detection thermocycler tomonitor the fluorescence in real-time, we managed to precisely control theinitiation of the helicase reaction through temperature tuning. Therefore, wewere able to demonstrate that this setup could provide a qualitative and aquantitative evaluation of the helicase domain of the UPF1 helicase (UPF1-HD) andthat several fluorophores could be used in parallel during the same run.
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