Teaching PCR for Simultaneous Sensing of Gene Transcription and Downstream Metabolites by Cucurbit[8]uril-Mediated Intervention of Polymerase Activity.

ABSTRACT

The target of typical PCR analysis is restricted to nucleic acids. To this end, we report here a novel strategy to simultaneously detect genetic and metabolic markers using commercial PCR kits with cucurbit[8]urils (CB[8]) implemented to manipulate the activity of Taq DNA polymerase. CB[8] binds with the nonionic surfactants and displaces them from the polymerase surface, resulting in decreased enzyme activity. Meanwhile, the inhibited enzyme can be reversibly activated when spermine, the downstream metabolite of ornithine decarboxylase (ODC), is present in the sample, which competitively binds to CB[8] and recovers polymerase activity. CB[8] was implemented in conventional PCR kits not only to reduce false-positive results but also to extend the detection range of PCR technology. With this novel method to detect ODC in cell lysates containing both the nucleotides and intracellular metabolites, positive results were only observed in highly active HEK 293T cells, whereas silent cells treated with ODC inhibitor showed negative readouts, therefore providing a simple but elegant dual-modality PCR method for precision diagnosis.